Fusion molecule based on novel taa variant

ABSTRACT

This invention provides novel carbonic anhydrase (CAIX) nucleic acid and peptide sequences, as well as related methods and compositions, including anti-cancer immunogenic agent(s) (e.g. vaccines and chimeric molecules) that elicit an immune response specifically directed against cancer cells expressing a CAIX antigenic marker. The novel CAIX variant and related compositions are useful in a wide variety of treatment modalities including, but not limited to protein vaccination, DNA vaccination, and adoptive immunotherapy.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/740,099, filed Jan. 11, 2013, which is a continuation of U.S.application Ser. No. 12/520,084, filed Feb. 26, 2010, now U.S. Pat. No.8,378,084, which is a National Stage application under 35 U.S.C. §371 ofPCT/US/2007/088676, filed Dec. 21, 2007, which is an applicationclaiming benefit under 35 U.S.C. §119(e) of U.S. Provisional ApplicationSer. No. 60/876,863 filed on Dec. 22, 2006, each of which isincorporated herein by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTINGAPPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file079822-1002988-0184430US_SequenceListing.txt, created on Mar. 16, 2016,20,636 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention generally relates to the science of oncology andtumor associated antigens. More specifically, the invention providesnovel carbonic anhydrase IX (CAIX) nucleic acid and peptide sequences,as well as related compositions and methods.

BACKGROUND OF THE INVENTION

Renal cell carcinoma (RCC) affects approximately 39,000 Americans andcauses an estimated 13,000 deaths per year. Approximately one third ofRCC patients will have advanced disease at presentation, and one thirdof patients with localized disease will eventually progress tometastatic disease. Of late, much progress and intensive researchefforts have been made in RCC therapies. Metastatic RCC poses atherapeutic challenge because of its resistance to conventional modes oftherapy, such as chemotherapy and radiation therapy, and considerablehurdles to eliciting an effective immune response for eradication of theremaining cancer cells (owing, at least in part, to their high invasionand metastatic potential).

Recent advances in the better understanding of molecular pathogenesis ofRCC has led to the development of several novel agents and approachesincluding immunotherapy and gene therapy, which have been applied inclinical trials with RCC patients. However, the treatments for advancedstages of RCC patients have been modest despite the considerableresearch efforts in this direction and the majority of patientsultimately succumbed to the disease. The long-term survival for patientswith metastatic RCC remains only 10-20%.

Although the ability of cytotoxic T cell to recognizepost-translationally modified epitopes has been acknowledged for severalyears, few have exploited this observation in a targeted approach togenerate immune responses of therapeutic benefit. Therefore, the successof cancer immunotherapy has focused on the identification oftumor-associated antigen (TAAs), which ideally are expressed exclusivelyby the cancer cells and not by normal adult tissues.

An exemplary TAA is carbonic anhydrase IX (CAIX), a transmembrane enzymethat regulates intracellular and extracellular pH during periods ofhypoxia by catalyzing the interconversion between CO₂ and bicarbonate.This cell surface protein may also be involved in oncogenesis and tumorprogression, and is thought to play a role in regulating cellularproliferation and possibly cellular adhesion.

New markers that correlate with clinical outcome or identify patientswith potentially aggressive disease, such as the novel CAIX variantprovided herein, can dramatically improve the diagnosis and managementof RCC. The present application accordingly provides a CAIX variant withnovel cDNA and amino acidic sequences. These sequences differ from theCAIX sequences reported thus far (e.g., sequences in NCBI Gene/proteindatabase and UniProtKB/Swiss-Prot protein database). In addition, theinvention provides fusion/chimeric molecules (e.g., GMCSF-CAIXv) andantibodies based on this newly identified CAIX variant. The CAIXvnucleic acids, polypeptides, and related constructs find use in thetreatment and diagnosis of cancer.

SUMMARY OF THE INVENTION

In a first aspect, a polypeptide comprising SEQ ID NO: 1 or a portionthereof having at least 20 contiguous amino acids of SEQ ID NO:1 andincluding one or more of residues M33, G121, and S374 is provided. Alsoprovided is an isolated or recombinant nucleic acid comprising apolynucleotide sequence selected from the group consisting of (a) SEQ IDNO: 2;

(b) a polynucleotide sequence encoding a polypeptide comprising SEQ IDNO: 1; and, (c) a polynucleotide sequence comprising a fragment of (a)or (b), which fragment encodes at least 20 contiguous amino acids of SEQID NO:1 and comprises one or more of residues M33, G121 and S374.

A further embodiment of the present invention provides methods of aidingin a cancer prognosis. The method include the steps of (a) quantifyingexpressed novel CAIX variant, if any, present in one or more samplesderived from a subject diagnosed with cancer to produce quantified novelCAIX variant expression data, wherein the expressed novel CAIX variantcomprises SEQ ID NO: 1, a portion of SEQ ID NO:1 comprising one or moreof residues M33, G121 and S374, or a nucleic acid encoding the novelCAIX variant or portion thereof; and, (b) correlating the quantifiednovel CAIX variant expression data with a probability of a cancerprognosis for the subject. Cancers for which prognoses can be determinedusing the methods of the claimed invention include, but are not limitedto, renal clear cell carcinoma, cervical cancer, bladder cancer, andhypoxia-inducible cancer.

In an additional embodiment of the present invention, methods of aidingin a cancer prognosis comprising the steps of (a) quantifying expressednovel CAIX variant polypeptides, if any, present in one or more samplesderived from a subject diagnosed with a cancer that expresses CAIX toproduce quantified CAIX polypeptide expression data, wherein the samplesare derived from a tumor and/or a metastatic lesion derived from atumor; and, (b) correlating the quantified novel CAIX variantpolypeptide expression data with a probability of a cancer prognosis,wherein a quantification percentage of 85% stratifies the prognosis forthe subject. Optionally, the quantified CAIX expression data are in acomputer-readable form, and wherein (b) comprises operating aprogrammable computer that comprises at least one database and executingan algorithm that determines closeness-of-fit between thecomputer-readable quantified CAIX expression data and database entries,which entries correspond to clinical and/or pathological data for apopulation of cancer patients to thereby correlate the quantified CAIXexpression data with the probability of the cancer prognosis for thesubject.

The present invention also provides constructs comprising a portion ofnovel CAIX variant (SEQ ID NO: 1) coupled to a granulocyte macrophagecolony stimulating factor (GM-CSF) or other immuno-effector or cytokine,wherein the portion of novel CAIX variant comprises one or more ofresidues M33, G121 and/or S374. Optionally, the construct is part of acomposition having a pharmaceutically acceptable diluent, excipient,and/or adjuvant. Accordingly, a CAIXv according to the inventioncontains the substitutions D33M, D121G, or N364S with respect to theCAIX protein.

In a further aspect, the present invention provides nucleic acidsencoding a fusion protein comprising a portion of novel CAIX variant(SEQ ID NO: 1) attached to a granulocyte macrophage colony stimulatingfactor (GM-CSF) or a cytokine or immuno-effector, wherein the portion ofnovel CAIX variant comprises one or more of residues M33, G121 and S374.Optionally, the nucleic acid is provided as an expression cassette or ina vector. The present invention also includes host cells transfectedwith said nucleic acid.

In an additional embodiment, the present invention provides methods ofproducing an anti-tumor vaccine, including the steps of culturing a celltransfected with a nucleic acid encoding a cytokine-CAIX fusion protein,under conditions where said cell expresses the fusion protein; andrecovering said fusion protein.

In a further aspect, the present invention provides methods of inducingan immune response against a CAIX antigen. The method includes the stepsof activating a cell of the immune system with a construct comprising aportion of novel CAIX variant (which portion comprises one or more ofresidues M33, G121 and S374 of SEQ ID NO: 1) coupled to a cytokine,(e.g., granulocyte macrophage colony stimulating factor) or otherimmuno-effector, whereby said activating provides an immune responsedirected against the novel CAIX variant. The activating step cancomprise contacting an antigen presenting cell with said construct.Alternatively, activation of the immune system cell can be achieved byinjecting the construct into an animal (e.g., a mammal).

Suitable cytokines for use in any of the above embodiments are mammalian(preferably, human) cytokines, including but not limited to thoseselected from the group consisting of interferons (IFNs) (e.g., IFN-α,IFN-γ), interleukins 1 to 20 (e.g., IL-2, IL-4, IL-6), and TNF. It iscontemplated that such cytokines may be employed in constructs andmethods generally as exemplified further below with GMCSF.

In an additional embodiment of the present invention, methods ofinhibiting the proliferation or growth of a transformed cell that bearsa novel CAIX variant are provided. The methods include removing orobtaining an immune cell from a mammalian host; activating said immunecell by contacting said cell with a protein comprising the novel CAIXvariant (SEQ ID NO: 1 or a fragment thereof comprising one or more ofresidues M33, G121 and S374), wherein the novel CAIX variant or fragmentthereof is attached to a cytokine (e.g., granulocyte macrophage colonystimulating factor (GM-CSF) or a fragment thereof); optionally expandingthe activated cell; and infusing the activated cell into an organismcontaining a transformed cell bearing the novel CAIX variant, therebyinhibiting the growth of the transformed cell. The immune cell can be,for example, peripheral blood lymphocytes (PBLs) or tumor infiltratinglymphocytes (TILs) from the mammalian host; optionally, the immune cellthus obtained and activated is infused back into the source host. Immunecells that can be employed in the methods of the present inventioninclude, but are not limited to, dendritic cells, antigen presentingcells, B lymphocytes, T-cells, and a tumor infiltrating lymphocytes.

A further embodiment of the present invention provides methods oftreating an individual having a cancer characterized by an alteredexpression of CAIX or CAIXv. The methods include the steps of (a)sensitizing antigen presenting cells in vitro with asensitizing-effective amount of a chimeric fusion protein comprising thenovel CAIX variant attached to a granulocyte macrophage colonystimulating factor (GM-CSF) or other cytokine, wherein the novel CAIXvariant comprises SEQ ID NO: 1 or a fragment of at least 20 contiguousamino acids of SEQ ID NO:1 and includes one or more of residues M33,G121, and S374; and (b) administering to an individual having saidcancer or metastasis a therapeutically effective amount of thesensitized antigen presenting cells. The antigen presenting cells can beautologous to the individual, or are MHC matched allogenic dendriticcells. Any of a number of cells (e.g., peripheral blood lymphocytes,monocytes, fibroblasts, TILs, and/or dendritic cells) by contacting thecells with said chimeric fusion protein. In a preferred embodiment, thesensitizing step comprises transfecting dendritic cells or RCCs with anucleic acid encoding said chimeric fusion protein. The CAIXv cellsurface protein is thought to be involved in oncogenesis and tumorprogression, and in regulating cellular proliferation and possiblycellular adhesion. CAIX and CAIXv is inducible by hypoxia, a state foundin many cancers and accordingly all tumors can be targeted by themethods of the invention. I

As CAIX and CAIXv is inducible by hypoxia, a state found in manycancers, accordingly all tumors, (e.g., all solid tumors) can betargeted by the methods of the invention. In some embodiments of any ofthe above, the cancer is cervical cancer, bladder cancer, or renalcancer.

In another aspect, the invention provides antibodies which are specificto a CAIXv polypeptide. These antibodies typically would have at leastan affinity for a CAIXv of 10⁻⁵ M or, more preferably, at least of 10⁻⁶M or 10⁻⁷M. In some further embodiments, these antibodies bind to aportion of SEQ ID NO:1 comprising one, two or three of residues M33,G121 and S374. Preferably, these antibodies are selective in binding toa CAIXv comprising one or more of residues M33, G121 and S374, ascompared to the human CAIX protein of SEQ ID NO:3 or as disclosed inU.S. patent application Ser. No. 10/511,465 to Bui et al. and relatedPCT application PCT/US2003/11561. For instance, the antibody can beselective in binding to a CAIXv comprising one or more of residues M33,G121 and S374, as compared to a CAIX protein having the sequence of SEQID NO:3 or another amino acid sequence at those particular positions.The relative binding affinity for a selective antibody can be at least5-fold or 10-different. These antibodies are also contemplated for usein the methods according to the invention.

These and a variety of additional features of the present invention willbecome evident upon review of the following disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A schematic representation of a CAIXv construct according to theinvention. The figure shows an N-terminal CAIXv linked via a linker to acarboxy terminal GMCSF polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present application accordingly provides a CAIX variant with novelcDNA and amino acidic sequences. These sequences differ from the CAIXsequences reported thus far (e.g., sequences in NCBI Gene/proteindatabase and UniProtKB/Swiss-Prot protein database). The sequences comefrom fresh kidney cells obtained from human patients. In addition, theinvention provides fusion/chimeric molecules (e.g., GMCSF-CAIXv) basedon this newly identified CAIX variant. The invention also provides CAIXvnucleic acids, polypeptides, and related constructs which find use inthe treatment and diagnosis of cancer.

This invention provides a novel approach to the treatment of renal cellcarcinomas, cervical cancers, and bladder cancers. In particular thisinvention pertains to the discovery that a chimeric molecule comprisinga granulocyte macrophage colony stimulating factor (GM-CSF) attached toa CAIXv kidney cancer specific antigen provides a highly effective“vaccine” that raises an immune response directed against renal cellcancers. The chimeric molecule can be used as a traditional vaccine orin adoptive immunotherapeutic applications. Nucleic acids encoding aGM-CSF-CAIXv fusion protein can be used as naked DNA vaccines or totransfect cell in an adoptive immunotherapeutic treatment regimen.

Thus in one embodiment, this invention provides a construct comprising aCAIXv polypeptide attached to a granulocyte macrophage colonystimulating factor (GM-CSF) or other cytokine or immuno-effector. TheGM-CSF is preferably a human GM-CSF, or a biologically active fragmentand/or mutant thereof. Similarly the CAIXv polypeptide is a preferably ahuman

CAIXv polypeptide. In particularly preferred embodiments the CAIXvpolypeptide is covalently attached to the GM-CSF (directly or through alinker). A suitable linker is encoded by the nucleotide sequence AAGCTTwhich encodes -Lys-Leu-. In a particularly preferred embodiment theCAIXv and the GM-CSF are components of a fusion protein (chemicallyconstructed or recombinantly expressed. In such fusion proteins, theCAIXv polypeptide and the GM-CSF are directly joined, or morepreferably, joined by a peptide linker ranging in length from 2 to about50, more preferably from about 2 to about 20, and most preferably fromabout 2 to about 10 amino acids. One preferred peptide linker is-Lys-Leu-. A particularly preferred construct has a CAIXv of SEQ ID NO:1.

Suitable cytokines for use in any of the above embodiments are mammalian(preferably, human) cytokines, including but not limited to thoseselected from the group consisting of interferons (IFNs) (e.g., IFN-α,IFN-γ), interleukins 1 to 20 (e.g., IL-2, IL-4, IL-6), and TNF. It iscontemplated that such cytokines may be employed in constructs andmethods generally as exemplified further below with GMCSF. Accordingly,in some embodiments, the GM-CSF of a construct described below isreplaced by a cytokine selected from the group consisting of interferons(IFNs) (e.g., IFN-α, IFN-γ), interleukins 1 to 20 (e.g., IL-2, IL-4,IL-6), and TNF. In some embodiments, one or more cytokines are fused tothe CAIXv.

In another embodiment this invention provides a composition comprisingthe chimeric molecules described herein and a pharmaceuticallyacceptable diluent or excipient.

This invention also provides a nucleic acid (e.g. a DNA or an RNA)encoding a fusion protein comprising a CAIXv kidney cancer specificpolypeptide or antigen attached to a granulocyte macrophage colonystimulating factor (GM-CSF). The CAIXv can be an antigenic fragment orcancer-specific epitope of CAIXv. Similarly the GM-CSF is a preferably ahuman GM-CSF or a biologically active fragment thereof. In one preferredembodiment the nucleic acid encodes a fusion protein where the CAIXv andthe GM-CSF are directly joined, or more preferably, joined by a peptidelinker ranging in length from 2 to about 50, more preferably from about2 to about 20, and most preferably from about 2 to about 10 amino acids.In certain embodiments, the nucleic acid may preferably encode a linkerthat is -Arg-Arg-. One preferred nucleic acid is the nucleic acid of SEQID NO: 2. In some preferred embodiments, the nucleic acid is a nucleicacid that encodes the polypeptide of SEQ ID NO: 1. The nucleic acid ispreferably in an expression cassette and in certain embodiments, thenucleic acid is present in a vector (e.g. a baculoviral vector).

This invention also provides a host cell transfected with one or more ofthe nucleic acids described herein. The host cell is preferably aeukaryotic cell, and most preferably an insect cell.

This invention also provides methods of producing an anti-tumor vaccine.The methods preferably involve culturing a cell transfected with anucleic acid encoding a chimeric GM-CSF-CAIXv chimeric molecule underconditions where the nucleic expresses a CAIXv-GM-CSF fusion protein andrecovering said fusion protein. Again the cell is preferably aeukaryotic cell, more preferably an insect (e.g. an SF9) cell.

In another embodiment, this invention provides methods of inducing animmune response against the CAIXv kidney cancer-specific antigen, and/ora cell displaying the CAIXv kidney cancer-specific antigen, and/or anycancer cell that expresses a CAIXv antigen, and/or an antigencross-reactive with a CAIXv antigen. The methods involve activating acell of the immune system with a construct comprising a kidney cancerspecific antigen (CAIXv) attached to a granulocyte macrophage colonystimulating factor (GM-CSF) whereby the activating provides an immuneresponse directed against the CAIXv antigen. In some embodiments, theactivating comprises contacting an antigen presenting cell (e.g.monocyte, or dendritic cell) with the construct (chimeric molecule). Incertain embodiments, the activated cell is a cytotoxic T-lymphocyte(CTL), or a tumor infiltrating lymphocyte, etc. The activating can alsoinvolve contacting a peripheral blood lymphocyte (PBL) or a tumorinfiltrating lymphocyte (TIL) with the construct. The contacting cantake place in vivo, or ex vivo (e.g., in vitro). In various embodiments,the activating comprises loading an antigen presenting cell (APC) with apolypeptide comprising a CAIXv. The activation can also comprisetransfecting a cell (e.g., a PBL, an APC, a TIL, a renal cell carcinomatumor cell, etc.) with a nucleic acid encoding a GM-CSF-CAIXv fusionprotein. The method may further comprise infusing cells (e.g. cytotoxicT lymphocytes) back into the mammal.

In still another embodiment this invention provides a method ofinhibiting the proliferation or growth of a transformed (e.g.neoplastic) kidney cell. The method involves activating a cell of theimmune system with a construct comprising a kidney cancer specificantigen (CAIXv) attached to a granulocyte macrophage colony stimulatingfactor (GM-CSF) whereby the activating provides an immune responsedirected against the CAIXv antigen and the immune response inhibits thegrowth or proliferation of a transformed kidney cancer cell. Inpreferred embodiments, the transformed kidney cancer cell is a renalcell carcinoma cell (e.g. in a solid tumor, a disperse tumor, or ametastatic tumor). The activating can comprise contacting an antigenpresenting cell (e.g. a dendritic cell) with the construct. Theactivated cell can include, but is not limited to a cytotoxicT-lymphocyte (CTL) a tumor infiltrating lymphocyte (TIL), etc. Incertain embodiments, the activating comprises injecting (or otherwiseadministering) to a mammal one or more of the following: a polypeptidecomprising a GM-CSF-CAIXv fusion protein; dendritic cells pulsed with aGM-CSF-CAIXv fusion protein; a gene therapy construct (e.g. adenovirus,gutless-adenovirus, retrovirus, lentivirus, adeno-associated virus,vaccinia virus, simian virus 40, etc) comprising a nucleic acid encodinga GM-CSF-CAIXv fusion protein, a dendritic expressing a GM-CSF-CAIXvfusion protein, a tumor cell (e.g. RCC) expressing a

GM-CSF-CAIXv fusion protein, a fibroblast expressing a GM-CSF-CAIXvfusion protein, a GM-CSF-CAIXv naked DNA, a transfection reagent (e.g.cationic lipid, dendrimer, liposome, etc. containing or complexed with anucleic acid encoding a GM-CSF-CAIXv polypeptide. In a particularlypreferred embodiment, activating comprises activating isolated dendriticcells/PMBCs. In another embodiment, the activating comprises contacting(in vivo or ex vivo) a peripheral blood lymphocyte (PBL) or a tumorinfiltrating lymphocyte (TIL) with said construct. The peripheral bloodcells and/or dendritic cells and/or monocytes are preferably infusedinto the subject.

This invention also provides a method of inhibiting the proliferation orgrowth of a transformed renal carcinoma cell (RCC) that bears a CAIXvantigen. The method involves removing an immune cell from a mammalianhost; activating the immune cell by contacting the cell with a proteincomprising a renal cell carcinoma specific antigen (CAIXv) attached to agranulocyte macrophage colony stimulating factor (GM-CSF) or a fragmentthereof, optionally expanding the activated cell; and infusing theactivated cell into an organism containing a transformed renal cellcarcinoma bearing a CAIXv. In certain embodiments, the activatingcomprises contacting the cell with one or more of the following: apolypeptide comprising a GM-CSF-CAIXv fusion protein; dendritic cellspulsed with a GM-CSF-CAIXv fusion protein; a gene therapy construct(e.g. adenovirus, gutless-adenovirus, retrovirus, lantivirus,adeno-associated virus, vaccinia virus, simian virus 40, etc) comprisinga nucleic acid encoding a GM-CSF-CAIXv fusion protein, a dendriticexpressing a GM-CSF-CAIXv fusion protein, a tumor cell (e.g. RCC)expressing a GM-CSF-CAIXv fusion protein, a fibroblast expressing aGM-CSF-CAIXv fusion protein, a GM-CSF-CAIXv naked DNA, a transfectionreagent (e.g. cationic lipid, dendrimer, liposome, etc. containing orcomplexed with a nucleic acid encoding a GM-CSF-CAIXv polypeptide. In aparticularly preferred embodiment, activating comprises activatingisolated dendritic cells/PMBCs. In another embodiment, the activatingcomprises contacting (in vivo or ex vivo) a peripheral blood lymphocyte(PBL) or a tumor infiltrating lymphocyte (TIL) with said construct. Theperipheral blood cells and/or dendritic cells and/or monocytes arepreferably infused into the subject. The removing may comprise isolatingand culturing peripheral blood lymphocytes and/or monocytes, and/ordendritic cells from the mammalian host. The infusing may involveinfusing the cultured cells or activated cells produced using thecultured cells into the host from which the immune cell was removed.

In still another embodiment, this invention provides a method oftreating an individual having a renal cell cancer. The method involvessensitizing antigen presenting cells (e.g., PBMCs, dendritic cells,etc.) in vitro with a sensitizing-effective amount of a chimeric fusionprotein comprising a renal cell carcinoma specific antigen (CAIXv)attached to a granulocyte macrophage colony stimulating factor (GM-CSF);and administering to an individual having said renal cell cancer ormetastasis a therapeutically effective amount of the sensitized antigenpresenting cells. In particularly preferred embodiments, the antigenpresenting cells are autologous to the individual or allogenic withmatched MHC. In certain embodiments, the sensitizing involves contactingperipheral blood lymphocytes or monocytes or dendritic cells withCAIXv-GM-CSF fusion protein. In certain embodiments, the sensitizinginvolves contacting PBL, TIL, monocyte, dendritic cell with aCAIXv-GM-CSF polypeptide and/or transfecting dendritic cell, APC, RCC,fibroblasts, with a nucleic acid encoding the chimeric fusion protein.

In some embodiments according to the invention, the patient or subjectis a mammal selected from the group consisting of a human, a non-humanprimate, a rodent, a porcine, a largomorph, a canine, a feline, anequine, a porcine, and a bovine.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which, the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used inaccordance with the definitions set out below.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a surface”includes a combination of two or more surfaces; reference to “bacteria”includes mixtures of bacteria, and the like.

The terms “carbonic anhydrase IX” and “CAIX” are herein considered to besynonymous with “CA9”, “MN”, and “G250.” The G250 antigen has beensequenced and revealed by database analysis to be homologous to theMN/CAIX antigen, a tumor-associated antigen originally identified inHeLa cells (Pastorek et al. (1994) “Cloning and characterization of MN,a human tumor associated protein with a domain homologous to carbonicanhydrase and a putative helix-loop-helix DNA binding segment,” Oncogene9:2877-2888 and Oosterwijk et al. (1996) “Molecular characterization ofthe renal cell carcinoma associated antigen G250,” Proc Amer AssocCancer Res 37:461). This antigen (MN/CAIX/CA9/G250) is a plasma membraneglycoprotein with an apparent molecular weight of 54/58 kDa, detectablein several types of malignancies; e.g. cervical and ovarian cancer (Liaoet al. et al. (1994) “Identification of the MN antigen as a diagnosticbiomarker of cervical intraepithelial squamous and glandular neoplasiaand cervical carcinomas,” Am J Pathol 145:598-609), renal cancer(Oosterwijk et al. (1986) “Immunohistochemical analysis of monoclonalantibodies to renal antigens,” Am J Pathol 123:301-309), colorectalcancer (Saamio et al. (1997) “Immunohistochemical study of colorectaltumors for expression of a novel transmembrane carbonic anhydrase, MN/CAIX, with potential value as a marker of cell proliferation,” Am J Pathol153:279-285), esophageal cancer (Turner et al. (1997) “MN antigenexpression in normal, preneoplastic, and neoplastic esophagus: aclinicopathological study of a new cancer-associated biomarker,” HumanPathol 28:740-744), bladder cancer (Uemura et al. (1997) “Expression oftumor-associated antigen MN/G250 in urologic carcinoma: potentialtherapeutic target,” J Urol (Suppl) 157:377), but not in the normaltissues except alimentary tract, which indicates that the CAIX proteinis associated with tumorigenicity. Sequential analysis has demonstratedthat the gene (MN/CAIX/CA9/G250) is a novel member of the carbonicanhydrase (CA) family and MN/CADUG250 is considered to be the onlytumor-associated CA isoenzyme. See, e.g., U.S. Pat. No. 6,297,051,entitled “MN GENE AND PROTEIN” issued Oct. 2, 2001 to Zavada et al.,which is incorporated by reference in its entirety for all purposes.

Carbonic anhydrase IX (CAIX) is a cell surface tumor-associated antigenthat is highly expressed and used as a molecular signature for mostRCCs. As such, CAIX has been an attractive biomarker for the developmentof CAIX targeted therapeutic interventions and diagnostics. U.S. patentapplication Ser. No. 10/511,465 to Bui et al. and related PCTapplication PCT/US2003/11561, which are incorporated herein by referencein their entirety, provides methods of using CAIX as a molecular markerassociated with RCC disease progression and survival before. The levelof expression of the molecular biomarker, reflected by itsimmunochemical staining profile, correlated with response to treatment,clinical factors, pathological features and survival. In addition, U.S.patent application Ser. No. 09/783,708 to Belldegrun et al., alsoincorporated herein by reference in its entirety, describes methods fortreatment (e.g. mitigation of symptoms) of cancers that express CAIX, oran antigen cross-reactive with CAIX.

The cDNA and amino acidic sequences of CAIX as reported in NCBIGene/protein database (and used in laboratories around the world)stemmed from cancer cell lines propagated in cell cultures system. Incontrast, the novel CAIX variant provided herein were identifieddirectly from RCC tumor patients' tissues via RT-PCR and sequenceanalysis. Surprisingly, the CAIX sequence obtained from tissue differsfrom the CAIX sequences derived from cell culture system and reported inother laboratories. In particular, we have found there are two or threeamino acids that vary from all of the reported CAIX amino acidicsequences in NCBI gene/protein database and UniProtKB/Swiss-Prot proteinsequence database, respectively. As such, the novel CAIX sequencesprovided herein, which reflect the protein present in situ (e.g., inCAIX-expressing cancer tissues), provides novel biomarker that can beemployed for identifying, improving and managing the therapeuticoutcomes in patients having CAIX-expressing cancers, such as metastaticRCC patients.

The novel CAIXv sequences are as follows:

TABLE 1 CAIX Protein Sequence (SEQ ID NO: 1):MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLMPVHPQRLPRMQEDSPLGGGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVEAPGGPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAKQLHTLSDTLWGPGDSRLQLSFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAGDILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYR PAEVAETGA

TABLE 2 CAIXv cDNA Sequence (SEQ ID NO: 2):ATGGCTCCCCTGTGCCCCAGCCCCTGGCTCCCTCTGTTGATCCCGGCCCCTGCTCCAGGCCTCACTGTGCAACTGCTGCTGTCACTGCTGCTTCTGATGCCTGTCCATCCCCAGAGGTTGCCCCGGATGCAGGAGGATTCCCCCTTGGGAGGAGGCTCTTCTGGGGAAGATGACCCACTGGGCGAGGAGGATCTGCCCAGTGAAGAGGATTCGCCCAGAGAGGAGGATCCACCCGGAGAGGAGGATCTACCTGGAGAGGAGGATCTACCTGGAGAGGAGGATCTACCTGAAGTTAAGCCTAAATCAGAAGAAGAGGGCTCCCTGAAGTTAGAGGATCTACCTACCGTTGAGGCTCCTGGAGGTCCTCAAGAACCCCAGAATAATGCCCACAGGGACAAAGAAGGGGATGACCAGAGTCATTGGCGCTATGGAGGCGACCCGCCCTGGCCCCGGGTGTCCCCAGCCTGCGCGGGCCGCTTCCAGTCCCCGGTGGATATCCGCCCCCAGCTCGCCGCCTTCTGCCCGGCCCTGCGCCCCCTGGAACTCCTGGGCTTCCAGCTCCCGCCGCTCCCAGAACTGCGCCTGCGCAACAATGGCCACAGTGTGCAACTGACCCTGCCTCCTGGGCTAGAGATGGCTCTGGGTCCCGGGCGGGAGTACCGGGCTCTGCAGCTGCATCTGCACTGGGGGGCTGCAGGTCGTCCGGGCTCGGAGCACACTGTGGAAGGCCACCGTTTCCCTGCCGAGATCCACGTGGTTCACCTCAGCACCGCCTTTGCCAGAGTTGACGAGGCCTTGGGGCGCCCGGGAGGCCTGGCCGTGTTGGCCGCCTTTCTGGAGGAGGGCCCGGAAGAAAACAGTGCCTATGAGCAGTTGCTGTCTCGCTTGGAAGAAATCGCTGAGGAAGGCTCAGAGACTCAGGTCCCAGGACTGGACATATCTGCACTCCTGCCCTCTGACTTCAGCCGCTACTTCCAATATGAGGGGTCTCTGACTACACCGCCCTGTGCCCAGGGTGTCATCTGGACTGTGTTTAACCAGACAGTAATGCTGAGTGCTAAGCAGCTCCACACCCTCTCTGACACCCTGTGGGGACCCGGTGACTCTCGGCTACAACTGAGCTTCCGAGCGACGCAGCCTTTGAATGGGCGAGTGATTGAGGCCTCCTTCCCTGCTGGAGTGGACAGCAGTCCTCGGGCTGCTGAGCCAGTCCAGCTGAATTCCTGCCTGGCTGCTGGTGACATTCTAGCCCTGGTTTTTGGCCTCCTTTTTGCTGTCACCAGCGTCGCGTTCCTTGTGCAGATGAGAAGGCAGCACAGAAGGGGAACCAAAGGGGGTGTGAGCTACCGCCCAGCAGAGGTAGCCGAGACTGGAGCCTAG

The term “CAIX variant” or “CAIXv”references a polypeptide of SEQ IDNO:1 or a portion of the polypeptide or a portion thereof, wherein theportion comprises at least 20 contiguous amino acids of SEQ ID NO:1 andincludes one or more of residues M33, G121, and S374. In someembodiments, the polypeptide comprises the full polypeptide sequence ofSEQ ID NO:1. In other embodiments, the portion comprises at least 20,30, 40, 50, 60, 70, 100, 200 or 300 contiguous amino acids. In someembodiments, the portion comprises M33, G121, or S374.

A CAIXv-GM-CSF construct or GM-CSF-CAIXv construct reference a chimericmolecule comprising a CAIX variant attached to a granulocyte-macrophagecolony stimulating factor. The attachment may be a chemical conjugation(direct or through a linker) or the chimeric molecule can be a fusionprotein (recombinantly expressed or assembled by condensation of the twosubject molecules). Accordingly, the notation “CAIXv-GM-CSF” or“GM-CSF-CAIXv” encompasses embodiments where the CAIXv and the GM-CSFare attached terminally or to an internal site and contemplatesattachment of the CAIXv to either the amino or carboxyl terminus of theGM-CSF. In addition, the term CAIXv-GMCSF may encompass chimericmolecules comprising fragments of CAIXv wherein the CAIXv fragmentsretain the epitope recognized by antibodies that specifically targetrenal cell carcinomas bearing the CAIXv protein or antigen. Similarly,the term may encompass chimeric molecules comprising fragments of GM-CSFwhere the GM-CSF retain the biological activity of native GM-CSF (e.g.are recognized by receptors that recognize native GM-CSF and/or showsimilar mitogenic activity, etc.). In some embodiments, the GM-CSF ishuman GM-CSF. The above CAIXv polypeptides can be used in the methodsand constructs of the invention.

The term “CAIXv nucleic acid” references a nucleic acid which encodes aCAIXv polypeptide. In one embodiment, the CAIXv nucleic acid sequence isSEQ ID NO:2. A CAIXv nucleic acid can be or comprise a polynucleotidesequence selected from the group consisting of (a) SEQ ID NO: 2; (b) apolynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 1;and, (c) a polynucleotide sequence comprising a fragment of (a) or (b),which fragment encodes at least 20 contiguous amino acids of SEQ ID NO:1and comprises one or more of residues M33, G121 and S374. The aboveCAIXv nucleic acids are suitable for use in the methods and constructsof the invention.

“Renal cell carcinoma” or “RCC” refers to carcinoma of the renalparenchyma. RCC is also often identified as renal cancer,“hypemephroma”, or adenocarcinoma of the kidney. There are four maintypes of renal cell carcinoma, namely, clear cell type, granular celltype, mixed granular and clear cell type, and spindle cell type.

The terms “polypeptide”, “peptide”, and “protein” are usedinterchangeably herein to refer to a polymer of amino acids that arecovalently bound by peptide linkages. The terms “polypeptide”,“peptide”, and “protein” include glycoproteins as well asnon-glycoproteins.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111 :2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

The term “immune cell” refers to a cell that is capable ofparticipating, directly or indirectly, in an immune response. Immunecells include, but are not limited to T-cells, B-cells, dendritic cells,cytotoxic T-cells, tumor infiltrating lymphocytes, etc.

As used herein, the term “activating” (e.g. as in activating a cell oractivating an immune response) includes direct activation as by contactwith the construct or by indirect activation as by contact with theconstruct or antigenic fragment via an antigen presenting cell (e.g. adendritic cell).

A “fusion protein” refers to a polypeptide formed by the joining of twoor more polypeptides through a peptide bond formed between the aminoterminus of one polypeptide and the carboxyl terminus of anotherpolypeptide. The fusion protein may be formed by the chemical couplingof the constituent polypeptides, or it may be expressed as a singlepolypeptide from nucleic acid sequence encoding the single contiguousfusion protein. A single chain fusion protein is a fusion protein havinga single contiguous polypeptide backbone.

A “spacer” or “linker” as used in reference to a fusion protein refersto a peptide that joins the proteins comprising a fusion protein.Generally a spacer has no specific biological activity other than tojoin the proteins or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of aspacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity of the molecule.

A “spacer” or “linker” as used in reference to a chemically conjugatedchimeric molecule refers to any molecule that links/joins theconstituent molecules of the chemically conjugated chimeric molecule.

“Antibody” refers to a polypeptide substantially encoded by at least oneimmunoglobulin gene or fragments of at least one immunoglobulin gene,that can participate in specific binding with a ligand. The termincludes naturally-occurring forms, as well as fragments andderivatives. Fragments within the scope of the term as used hereininclude those produced by digestion with various peptidases, such asFab, Fab′ and F(ab)′2 fragments, those produced by chemicaldissociation, by chemical cleavage, and recombinantly, so long as thefragment remains capable of specific binding to a target molecule, suchas a host cell protein. Typical recombinant fragments, as are produced,e.g., by phage display, include single chain Fab and scFv (“single chainvariable region”) fragments. Derivatives within the scope of the terminclude antibodies (or fragments thereof) that have been modified insequence, but remain capable of specific binding to a target molecule,including interspecies chimeric and humanized antibodies. As usedherein, antibodies can be produced by any known technique, includingharvest from cell culture of native B lymphocytes, hybridomas,recombinant expression systems, by phage display, or the like.

Accordingly, antibodies comprise a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)I by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

Accordingly, the term antibody also embraces minibodies, diabodies,triabodies and the like. Diabodies are small bivalent biospecificantibody fragments with high avidity and specificity. Their high signalto noise ratio is typically better due to a better specificity and fastblood clearance increasing their potential for diagnostic andtherapeutic targeting of specific antigen (Sundaresan et al., J Nucl Med44:1962-9 (2003). In addition, these antibodies are advantageous becausethey can be engineered if necessary as different types of antibodyfragments ranging from a small single chain Fv to an intact IgG withvarying isoforms (Wu & Senter, Nat. Biotechnol. 23:1137-1146 (2005)). Insome embodiments, the antibody fragment is part of a diabody. Exemplarydiabodies for use according to the invention include those designatedherein as KS41, KS49, KS83, KS89.

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many techniques known in the art can be used(see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3^(rd) ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No.4,816,567) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized or human antibodies (see,e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992);Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996);Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980 , WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as import residues,which are typically taken from an import variable domain. Humanizationcan be essentially performed following the method of Winter andco-workers (see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies can be selectedto obtain only those polyclonal antibodies that are specificallyimmunoreactive with the selected antigen and not with other proteins.This selection may be achieved by subtracting out antibodies thatcross-react with other molecules. A variety of immunoassay formats maybe used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual(1998) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

“Prognosis” refers to a forecast as to the probable outcome of a diseasestate a determination of the prospect as to recovery from a disease asindicated by the nature and symptoms of a case, the monitoring of thedisease status of a patient, the monitoring of a patient for recurrenceof disease, and/or the determination of the preferred therapeuticregimen for a patient.

“Quantification percentage” refers to a CAIX expression score thatincludes the percentage of a sample (e.g., a target tissue or cellularsample, such as a sample from a renal tumor, a sample from a metastaticlesion derived from a metastatic lesion, and/or the like) that haspositive CAIX expression. In preferred embodiments, the quantificationpercentage of a sample refers a CAIX expression score that includes theextent of staining or staining percentage (e.g., the percentage of cellsin a sample that stain positively for CAIX, etc.). In certainembodiments, other factors such as staining intensity and the percentagestaining at maximal staining intensity are also included in a CAIXexpression score for a particular sample. For example, as illustrated inan example provided below, survival tree analysis of CAIX scoringinformation from the analyzed tissue arrays identified that a stainingpercentage of 85% was an ideal cutoff for stratification for patientsurvival. Staining percentages >85%, irrespective of intensity, wereconsidered high CAIX staining, whereas those ≦85% were considered lowCAIX staining.

As noted above, we have identified a CAIX variant with novel cDNA andamino acidic sequences, using polymerase chain reaction (PCR) cloningmethods and sequence analysis on samples from RCC patient tissues. Thenucleic acid and protein sequence of the novel CAIX variant(alternatively referred to herein as the “mutant CAIX”) differ from theCAIX sequences reported thus far (e.g., sequences in NCBI Gene/proteindatabase and UniProtKB/Swiss-Prot protein database). In addition,fusion/chimeric molecules (GMCSF-CAIX) based on this newly identifiedCAIX variant have been constructed. The constructs are to be used, atleast initially, for targeted kidney cancer vaccine therapy, based uponearly laboratory results as well as the fact that GMCSF gene/vaccinetherapy has been shown to have clinical anti-tumor activity in RCC.

TARGETED CANCER VACCINE THERAPY

As noted above, a CAIX variant with novel cDNA and amino acidicsequences from RCC patient tissues (and having entirely differentsequences from those reported previously) is provided in the presentinvention. Among other advantages, the novel CAIX sequence can be usedto generate more effective targeted vaccines for a variety of cancersthat express CAIX on the cell surface.

For example, as a part of the research program of targeted kidney cancervaccine therapy, a fusion/chimeric molecules (GMCSF-CAIX) based on thenewly identified CAIX variant was constructed. The rationale for thisapproach is based on early laboratory results, as well as the fact thatcytokine-gene/vaccine therapy has previously been shown to haveanti-tumor activity in clinical trials. The novel human RCC CAIX variantwas combined with GMCSF, a potent cytokine, to significantly enhanceanti-cancer efficacy of CAIX-based vaccine.

The present invention provides fusion molecules of GMCSF-CAIXv, as wellas GMCSF-CAIXv Adenoviral Vector Gene Delivery Systems. In a preferredembodiment, the pAd-CMV shuttle vector in current construction ofGMCSF-CAIXv was utilized to replace GFP-containing padTrack-CMV shuttlevector reported previously in our laboratory. In addition, ananotechnology platform combined with sustained/controlled deliverytechnology can be used to achieve longer-term expression of GMCSF-CAIXvfusion molecule, e.g., in dendritic cells.

Renal Cell Carcinoma Treatments

In view of the well recognized important of CAIX in RCC biology, theCAIX variant with novel cDNA/amino acidic sequences provided hereinprovides a new target for the development of new, specific strategies toaugment anti-cancer efficacy, as well as for RCC disease progression andsurvival. Both the novel CAIXv protein and monoclonal antibodiesdirected thereto may be developed as therapeutics and diagnostics forCAIXv-expressing cancers. The novel CAIX variant can also be employed instrategies that target the signaling pathways regulated by CAIX.

METHODS FOR PROGNOSIS

The novel CAIX variant is also useful as a prognostic biomarker andpathway signature to evaluate the relative expression level of CAIX incancerous tissue. For example, CAIX has been employed in the evaluationof RCC patients via collection of data from immunohistochemicalanalysis, RT-PCR and western blot, which were compared with measuredexpression levels in autologous normal kidney tissue, or with normalexpression level of control tissue. The novel CAIX variant of thepresent invention provides an improved prognostic biomarker, and can beused in the methods described in U.S. Ser. No. 10/511,465 to Bui et al.and related PCT application PCT/US2003/11561, to provide prognosticinformation for patients afflicted with various CAIX-expressing cancers,in addition to renal cell carcinoma (RCC). The methods includequantifying CAIX. In addition to reliably predicting clinical outcome,the methods of the present invention also can be used to better identifyhigh-risk patients in need of adjuvant immunotherapy and/orCAIX-targeted therapies, among other courses of treatment.

CHIMERIC CONSTRUCTS

As noted above, the invention provides a novel approach to the treatment(e.g. mitigation of symptoms) of any type of cancer that expresses thenovel CAIX variant or an antigen cross-reactive with CAIXv or CAIX (e.g.renal cell carcinoma, cervical cancer, bladder cancer, hypoxia-induciblecancers, and the like. In one embodiment of the invention, a chimericmolecule comprising the novel CAIX variant attached to agranulocyte-macrophage colony stimulating factor (GM-CSF) is provided.The chimeric molecule can be administered to a patient (e.g., byvaccination), leading to activation of antigen presenting cells (e.g.,dendritic cells). Presentation of the CAIXv antigen on HLA class I thenactivates CAIX-specific cytotoxic T cells, which can then lyseCAIX-positive cancer cells. In addition, or alternatively, the CAIXvpeptide is presented on HLA class II cells, resulting in activation ofCAIX-specific T helper cells, which then activate or maintain thekilling activity of CTLs.

Details with respect to the preparation and administration of chimericconstructs can be found, for example, in U.S. patent application Ser.No. 09/783,708 to Belldegrun et al., the contents of which are herebyincorporated by reference in their entirety.

This invention provides a novel approach to the treatment (e.g.mitigation of symptoms) of a renal cell carcinoma or any type of cancerthat expresses CAIX, including particularly CAIXv. In particular thisinvention utilizes a chimeric molecule comprising a CAIXv attached to agranulocyte-macrophage colony stimulating factor (GM-CSF). In oneembodiment, the GM-CSF has the sequence of SEQ ID NO:4. Without beingbound to a particular theory, it is believed that this chimeric moleculeaffords two modes of activity. Vaccination of patients with advancedrenal cell carcinoma using a chimeric CAIXv-GM-CSF molecule will resultin activation of the patient's dendritic cells (DC), the most potentantigen presenting cells. The dendritic cells take up GM-CSF, e.g., viathe GM-CSF receptor and the attached CAIXv is co-transported by virtueof its attachment to the GM-CSF. The dendritic cells process the CAIXvnand present CAIXv peptides on HLA class I which then activates CAIXvspecific cytotoxic T cells (CD3⁺CD8⁺) which can then lyse CAIXv positivekidney cancer cells. In addition, or alternatively, the CAIXv peptide ispresented on HLA class II cells that activate CAIXv specific T helpercells which then activate or maintain the killing activity of CTLs.

In certain embodiments, a nucleic acid encoding a CAIXv-GM-CSF constructcan be administered as a “naked DNA” vaccine. In this approach, theorganism/patient is injected, e.g. intramuscularly, with a nucleic acidencoding a CAIXv-GM-CSF fusion protein. The nucleic acid is expressedwithin the organism leading to the production of a CAIXv-GM-CSF fusionprotein which then elicits an anti-renal cell carcinoma immune responseas described above.

In another embodiment, the chimeric CAIXv-GM-CSF molecules can be usedin adoptive immunotherapy. In this instance, the chimeric molecule(fusion protein) or a nucleic acid encoding the chimeric molecule isused to activate lymphocytes (e.g. T-cells) ex vivo. The activatedlymphocytes are optionally expanded, ex vivo, and then re-infused backinto the subject (patient) where they specifically attack and lyse CAIXvpositive tumor cells (e.g. kidney cells tumor or cervical cancer cells).

In particularly preferred embodiments, this invention utilizes one ormore of the following formulations:

1. A polypeptide comprising a fusion protein of GM-CSF (e.g., a GM-CSFpolypeptide of SEQ ID NO:4) and a CAIXv polypeptide.

2. Dendritic, or other cells, pulsed with a polypeptide comprisingGM-CSF and CAIXv as a fusion protein;

3. Nucleic acids encoding a fusion protein of GM-CSF and CAIXv in a“gene therapy” vector (e.g. adenovirus, gutless-adenovirus, retrovirus,lantivirus, adeno-associated virus, vaccinia virus, simian virus 40,etc.)

4. Dendritic cells transfected with a nucleic acid encoding a fusionprotein of GM-CSF and CAIXv (e.g., via recombinant virus, plasmid DNAtransfection, and the like);

5. Tumor cells (e.g. RCC cells) comprising a nucleic acid encoding afusion protein of GM-CSF and CAIXv;

6. A nucleic acid encoding a fusion protein of GM-CSF and CAIXv (e.g.“naked DNA”); and

7. A nucleic acid encoding a fusion protein of GM-CSF and CAIXvcomplexed with a transfection agent (e.g., DMRIE/DOPE lipid, dendrimers,etc.).

Each of these formulations can be directly administered to an organism(e.g. a mammal having a cancer that expresses CAIXv, or an antigencross-reactive to a CAIXv antigen) or can be used in an adoptiveimmunotherapy context. In the latter approach, the adoptiveimmunotherapy preferably utilizes cells derived from peripheral blood(e.g. peripheral blood lymphocytes (PBLs) or cells derived from a tumor(e.g. tumor infiltrating lymphocytes. (TILs)). Administration of theformulation results in activation and propagation of CAIXv-targetedcytotoxic T cells in PBMC or TIL cultures. Infusion of theCAIXv-targeted CTLs into the patient results in the development andmaintenance of a CAIXv-directed immune response.

The formulations identified above can also be administered directly to amammal for “in vivo” vaccination. Thus, for example, GM-CSF-CAIXvpolypeptides or nucleic acids endoding such polypeptides can beadministered to the organism as “traditional” vaccines. The otherimmunogenic formulations identified above, however, are also highlyactive in vivo and can also be “directly” administered to an organism asa “vaccine”. Thus, for example, dendritic cells pulsed with aGM-CSF-CAIXv fusion protein, dendritic, or other cells, transfected witha nucleic acid encoding a GM-CSF-CAIXv fusion protein, gene therapyvectors encoding a GM-CSF-CAIXv polypeptide, can all be administered toan organism where they induce and maintain a population ofCAIXv-directed cytotoxic T cells.

CAIXv-GM-CSF chimeric molecules e.g. when used in vivo as a vaccine orin an adoptive immunotherapeutic modality can induce a highly vigorousimmune response specifically directed at renal cell carcinomas. Theapproach results in the death or inhibition of neoplastic renal cellswhether diffuse (e.g. motile metastatic cells) or aggregated (e.g. as ina solid tumor). These methods can accompany administration of otheragents (e.g. immunomodulatory or cytotoxic agents, such as cytokines ordrugs).

It is recognized that the methods of this invention need not showcomplete tumor elimination (e.g. a “cure”) to be of value. Even a slightdecrease in the growth rate of a tumor, and/or in the propagation ofmetastatic, or other neoplastic, cells can be clinically relevantimproving the quality and/or duration of life. Of course, given the highefficacy observed, it is expected that the methods of this invention mayoffer a significant or complete degree of remission particularly whenused in combination with other treatment modalities (e.g. surgery,chemotherapy, interleukin therapy, TGFβ or IL-10 antisense therapy,etc.).

I. CAIXv-GMCSF Chimeric Molecules and Their Expression.

This invention utilizes a chimeric molecule comprising a CAIXv kidneycancer-specific antigen attached to a granulocyte-macrophage colonystimulating factor (GM-CSF/GMCSF). to induce a cell-mediated immuneresponse targeted to renal tumor cells. In a chimeric molecule, two ormore molecules that exist separately in their native state are joinedtogether to form a single molecule having the desired functionality ofall of its constituent molecules. In this instance, the constituentmolecules are CAIXv and GM-CSF respectively. The CAIXv provides anepitope that is presented (e.g. to T-cells) resulting in activation andexpansion of those cells and the formation of cytotoxic cells (e.g.cytotoxic T lymphocytes, tumor infiltrating lymphocytes (TILs), etc.)that are direct to tumor cells bearing the CAIXv or a CAIXv antigen. TheGM-CSF acts both to stimulate components of the immune system (e.g.monocytes, dendritic cells, NK, PMN, PBMC, etc.) and to mediate uptakeof the associated CAIXv or CAIXv antigen by dendritic cells. Inaddition, particularly in adoptive immunotherapeutic modalities, theGM-CSF also can act as an adjuvant.

The attachment of the CAIXv to the GM-CSF can be direct (e.g. a covalentbond) or indirect (e.g. through a linker). In addition, the CAIXv andthe GM-CSF proteins can be attached by chemical modification of theproteins or they can be expressed as a recombinant fusion protein.Detailed methods of producing the individual components and the chimericmolecule are provided below.

The nucleic acid sequence of CAIXv is disclosed herein. The nucleic acidsequence of GM-CSF (e.g. human GM-CSF) is well known to those of skillin the art (see, e.g., GenBank accession no: E02287).

Using this sequence information nucleic acids encoding CAIXv, GM-CSF, ora chimeric CAIXv-GM-CSF can be produced using standard methods wellknown to those of skill in the art. For example, the nucleic acid(s) maybe cloned, or amplified by in vitro methods, such as the polymerasechain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR), etc. A wide variety of cloning and invitro amplification methodologies are well known to persons of skill inthe art.

Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashionet al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, Sambrook, and Ausubel,as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR ProtocolsA Guide to Methods and Applications (Innis et al. eds) Academic PressInc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990)C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94; (Kwoh et al.(1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc.Natl. Acad. Sci. USA 87, 1874; Lomeli et al. (1989) J. Clin. Chem., 35:1826; Landegren et al., (1988) Science, 241: 1077-1080; Van Brunt (1990)Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene, 4: 560; andBarringer et al. (1990) Gene, 89: 117.

The CAIXv and the GM-CSF molecules of a CAIXv-GMCSF construct may bejoined together in any order. Thus, the CAIXv can be joined to eitherthe amino or carboxy termini of the GM-CSF. Where the molecules arechemically conjugated, they need not be joined end to end and can beattached at any convenient terminal or internal site.

The CAIXv and GM-CSF may be attached by any of a number of means wellknown to those of skill in the art. Typically the CAIXv and the GM-CSFare conjugated, either directly or through a linker (spacer). Becauseboth molecules are polypeptides, in one embodiment, it is preferable torecombinantly express the chimeric molecule as a single-chain fusionprotein that optionally contains a peptide spacer between the GM-CSF andthe CAIXv.

Means of chemically conjugating molecules are well known to those ofskill. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on an effectormolecule to bind the effector thereto.

A “linker”, as used herein, is a molecule that is used to join the CAIXvto the GM-CSF. In preferred embodiments, the linker is capable offorming covalent bonds to both the CAIXv and GM-CSF. Suitable linkersare well known to those of skill in the art and include, but are notlimited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. In certain embodiments, the linkersmay be joined to amino acids comprising CAIXv and/or GM-CSF throughtheir side groups (e.g., through a disulfide linkage to cysteine).However, in a preferred embodiment, the linkers will be joined to thealpha carbon amino and carboxyl groups of the terminal amino acids. Thelinker may be bifunctional, having one functional group reactive with asubstituent on the CAIXv and a different functional group reactive witha substituent on the GM-CSF. Alternatively, the CAIXv and/or the GM-CSFmay be derivatized to react with a “mono-functional” linker (see, e.g.,U.S. Pat. Nos. 4,671,958 and 4,659,839 for procedures to generatereactive groups on peptides).

In a particularly preferred embodiment, the chimeric molecules of thisinvention are fusion proteins. The fusion protein can be chemicallysynthesized using standard chemical peptide synthesis techniques, or,more preferably, recombinantly expressed. Where both molecules arerelatively short the chimeric molecule may be synthesized as a singlecontiguous polypeptide. Solid phase synthesis in which the C-terminalamino acid of the sequence is attached to an insoluble support followedby sequential addition of the remaining amino acids in the sequence is apreferred method for the chemical synthesis of the polypeptides of thisinvention. Techniques for solid phase synthesis are described by Baranyand Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nded. Pierce Chem. Co., Rockford, Ill. (1984).

In a most preferred embodiment, the chimeric fusion proteins of thepresent invention are synthesized using recombinant DNA methodology.Generally this involves creating a DNA sequence that encodes the fusionprotein, placing the DNA in an expression cassette under the control ofa particular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

In one embodiment, accordingly, the invention provides a nucleic acidwhich encodes a fusion of hGMCSF with hCAIXv of the sequence (SEQ IDNO:7):

(hGMCSF)-(linker)-(CAIXv):ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGACTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAG AAGCTTATGGCTCCCCTGTGCCCCAGCCCCTGGCTCCCTCTGTTGATCCCGGCCCCTGCTCCAGGCCTCACTGTGCAACTGCTGCTGTCACTGCTGCTTCTGATGCCTGTCCATCCCCAGAGGTTGCCCCGGATGCAGGAGGATTCCCCCTTGGGAGGAGGCTCTTCTGGGGAAGATGACCCACTGGGCGAGGAGGATCTGCCCAGTGAAGAGGATTCGCCCAGAGAGGAGGATCCACCCGGAGAGGAGGATCTACCTGGAGAGGAGGATCTACCTGGAGAGGAGGATCTACCTGAAGTTAAGCCTAAATCAGAAGAAGAGGGCTCCCTGAAGTTAGAGGATCTACCTACCGTTGAGGCTCCTGGAGGTCCTCAAGAACCCCAGAATAATGCCCACAGGGACAAAGAAGGGGATGACCAGAGTCATTGGCGCTATGGAGGCGACCCGCCCTGGCCCCGGGTGTCCCCAGCCTGCGCGGGCCGCTTCCAGTCCCCGGTGGATATCCGCCCCCAGCTCGCCGCCTTCTGCCCGGCCCTGCGCCCCCTGGAACTCCTGGGCTTCCAGCTCCCGCCGCTCCCAGAACTGCGCCTGCGCAACAATGGCCACAGTGTGCAACTGACCCTGCCTCCTGGGCTAGAGATGGCTCTGGGTCCCGGGCGGGAGTACCGGGCTCTGCAGCTGCATCTGCACTGGGGGGCTGCAGGTCGTCCGGGCTCGGAGCACACTGTGGAAGGCCACCGTTTCCCTGCCGAGATCCACGTGGTTCACCTCAGCACCGCCTTTGCCAGAGTTGACGAGGCCTTGGGGCGCCCGGGAGGCCTGGCCGTGTTGGCCGCCTTTCTGGAGGAGGGCCCGGAAGAAAACAGTGCCTATGAGCAGTTGCTGTCTCGCTTGGAAGAAATCGCTGAGGAAGGCTCAGAGACTCAGGTCCCAGGACTGGACATATCTGCACTCCTGCCCTCTGACTTCAGCCGCTACTTCCAATATGAGGGGTCTCTGACTACACCGCCCTGTGCCCAGGGTGTCATCTGGACTGTGTTTAACCAGACAGTAATGCTGAGTGCTAAGCAGCTCCACACCCTCTCTGACACCCTGTGGGGACCCGGTGACTCTCGGCTACAACTGAGCTTCCGAGCGACGCAGCCTTTGAATGGGCGAGTGATTGAGGCCTCCTTCCCTGCTGGAGTGGACAGCAGTCCTCGGGCTGCTGAGCCAGTCCAGCTGAATTCCTGCCTGGCTGCTGGTGACATTCTAGCCCTGGTTTTTGGCCTCCTTTTTGCTGTCACCAGCGTCGCGTTCCTTGTGCAGATGAGAAGGCAGCACAGAAGGGGAACCAAAGGGGGTGTGAGCTACCGCCCAGCAGAGGTAGCCGAGACTGGAGCCTAG

In another embodiment, the invention provides a nucleic acid sequenceencoding a fusion protein (SEQ ID NO:8) of the human GMCSF proteinsequence and the CAIXv protein sequence joined by a polypeptide linker(e.g., -Lys-Leu-).

hGMCSF MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQTITFESFKENLKDFLLVIPFDCWEP VQElinker (e.g., -Lys-Leu-) CAIXv proteinMAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLMPVHPQRLPRMQEDSPLGGGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVEAPGGPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAKQLHTLSDTLWGPGDSRLQLSFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAGDILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYRPAEVAETGA

DNA encoding the fusion protein of this invention (GM-CSF-CAIXv) may beprepared by any suitable method, including, for example, cloning andrestriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et at Meth.Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al.,Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett., 22: 1859-1862 (1981); and the solidsupport method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In a preferred embodiment, DNA encoding fusion proteins of the presentinvention is using DNA amplification methods such as polymerase chainreaction (PCR).

The nucleic acid constructs can have a linker (gcggcg) between thenucleic acids encoding CAIXvand GM-CSF. The linker sequence is used toseparate GM-CSF and CAIXv by a distance sufficient to ensure that, in apreferred embodiment, each domain properly folds into its secondary andtertiary structures. Preferred peptide linker sequences adopt a flexibleextended conformation, do not exhibit a propensity for developing anordered secondary structure that could interact with the functionalGM-CSF and CAIXv domains. Typical amino acids in flexible proteinregions include Gly, Asn and Ser. Virtually any permutation of aminoacid sequences containing Gly, Asn and Ser would be expected to satisfythe above criteria for a linker sequence. Other near neutral aminoacids, such as Thr and Ala, also may be used in the linker sequence.Thus, amino acid sequences useful as linkers of GM-CSF and CAIXv includethe Gly₄SerGly₅Ser linker (SEQ ID NO:5) used in U.S. Pat. No. 5,108,910or a series of four (Ala Gly Ser) residues (SEQ ID NO:6), etc. Stillother amino acid sequences that may be used as linkers are disclosed inMaratea et al. (1985), Gene 40: 39-46; Murphy et al. (1986) Proc. Nat'l.Acad. Sci. USA 83: 8258-62; U.S. Pat. No. 4,935,233; and U.S. Pat. No.4,751,180.

The length of the peptide linker sequence may vary without significantlyaffecting the biological activity of the fusion protein. In onepreferred embodiment of the present invention, a peptide linker sequencelength of about 2 amino acids is used to provide a suitable separationof functional protein domains, although longer linker sequences also maybe used. The linker sequence may be from 1 to 50 amino acids in length.In the most preferred aspects of the present invention, the linkersequence is from about 1-20 amino acids in length. In the specificembodiments disclosed herein, the linker sequence is from about 2 toabout 15 amino acids, and is advantageously from about 2 to about 10amino acids. Peptide linker sequences not necessarily required in thefusion proteins of this invention.

Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

Where it is desired to recombinantly express either the CAIXv, theGM-CSF, or the CAIXv-GM-CSF fusion protein, the nucleic acid sequencesencoding the desired protein are typically operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements typically include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences that control the terminationof transcription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants may additionally beincorporated.

The nucleic acid sequences encoding the fusion proteins may be expressedin a variety of host cells, including E. coli and other bacterial hosts,and eukaryotic host cells including but not limited to yeast, insectcells (e.g. SF9 cells) and various other eukaryotic cells such as theCOS, CHO and HeLa cells lines and myeloma cell lines. The recombinantprotein gene will be operably linked to appropriate expression controlsequences for each host. For E. coli this includes a promoter such asthe T7, trp, or lambda promoters, a ribosome binding site and preferablya transcription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences. In one particularly preferred embodiment the GM-CSF-CAIXvfusion gene is inserted into polyhedrin gene locus-based baculovirustransfer vector (e.g., pVL 1393, available from PharMingen) andexpressed in insect cells (e.g. SF9 cells).

In view of the redundancy of the genetic code, in any of the embodimentsusing a nucleic acid according to the invention, the sequence of thenucleic acid encoding the protein can optionally be optimized forexpression in bacteria, human cells, mammalian cells, insect cells or invitro translation as known to one of ordinary skill in the art.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo, and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, his tag capture, affinity columns, column chromatography,gel electrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the CAIXv, GM-CSF, orGM-CSF-CAIXv protein may possess a conformation substantially differentthan the native conformations of the polypeptide(s). In this case, itmay be necessary to denature and reduce the polypeptide and then tocause the polypeptide to re-fold into the preferred conformation.Methods of reducing and denaturing proteins and inducing re-folding arewell known to those of skill in the art (See, Debinski et al. (1993) J.Biol. Chem., 268: 14065-14070; Kreitman and Pastan, (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205:263-270). Debinski et al., for example, describe the denaturation andreduction of inclusion body proteins in guanidine-DTE. The protein isthen refolded in a redox buffer containing oxidized glutathione andL-arginine.

One of skill would recognize that modifications can be made to theGM-CSF, CAIXv, or GM-CSF-CAIXv proteins without diminishing theirbiological activity. Some modifications may be made to facilitate thecloning, expression, or incorporation of the constituent molecules intoa fusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids placedon either terminus to create conveniently located restriction sites ortermination codons, or to simplify purification such as polyhistidinetag sequence.

II. In Vivo Protein Vaccination.

Immunogenic compositions (e.g. vaccines) are preferably prepared fromthe CAIXv-GM-CSF fusion proteins of this invention. The immunogeniccompositions including vaccines may be prepared as injectables, asliquid solutions, suspensions or emulsions. The active immunogenicingredient or ingredients may be mixed with pharmaceutically acceptableexcipients which are compatible therewith. Such excipients are wellknown to those of skill in the art and include, but are not limited towater, saline, dextrose, glycerol, ethanol, and combinations thereof.The immunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof.

The immunogenic CAIXv-GM-CSF compositions may be administeredparenterally, by injection subcutaneous, intravenous, intradermal,intratumoral, or intramuscularly injection. Alternatively, theimmunogenic compositions formed according to the present invention, maybe formulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Such suppositories may be formed from mixtures containingthe active immunogenic ingredient (s) in the range of about 0.5 to about10%, preferably about 1 to 2%. Oral formulations may include normallyemployed carriers such as, pharmaceutical grades of saccharine,cellulose and magnesium carbonate. These compositions can take the formof solutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain about 1 to 95% of the activeingredient(s), preferably about 20 to about 75%.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, immunogenic and protective. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizeantibodies, and if needed, to produce a cell-mediated immune response.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges arereadily determinable by one skilled in the art and may be of the orderof micrograms to milligrams of the active ingredient(s) per vaccination.The antigenic preparations of this invention can be administered byeither single or multiple dosages of an effective amount. Effectiveamounts of the compositions of the invention can vary from 0.01-1,000microgram/ml per dose, more preferably 0.1-500 microgram/ml per dose,and most preferably 10-300 microgram/ml per dose.

Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent booster administrations. The dosage may also depend or theroute of administration and will vary according to the size of the host.

The concentration of the active ingredient (chimeric protein) in animmunogenic composition according to the invention is in general about 1to 95%.

Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants. While the GM-CSF component of thechimeric molecule can, itself act as an adjuvant, other adjuvants can beused as well. Adjuvants enhance the immunogenicity of an antigen but arenot necessarily immunogenic themselves. Adjuvants may act by retainingthe antigen locally near the site of administration to produce a depoteffect facilitating a slow, sustained release of antigen to cells of theimmune system. Adjuvants can also attract cells of the immune system toan antigen depot and stimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Intrinsicadjuvants, such as lipopolysaccharides, normally are the components ofthe killed or attenuated bacteria used as vaccines. Extrinsic adjuvantsare immunomodulators which are formulated to enhance the host immuneresponses. Thus, adjuvants have been identified that enhance the immuneresponse to antigens delivered parenterally. Some of these adjuvants aretoxic, however, and can cause undesirable side-effects, making themunsuitable for use in humans and many animals. Indeed, only aluminumhydroxide and aluminum phosphate (collectively commonly referred to asalum) are routinely used as adjuvants in human and veterinary vaccines.The efficacy of alum in increasing antibody responses to diphtheria andtetanus toxoids is well established and a HBsAg vaccine has beenadjuvanted with alum.

A wide range of extrinsic adjuvants can provoke potent immune responsesto antigens. These include saponins complexed to membrane proteinantigens (immune stimulating complexes), pluronic polymers with mineraloil, killed mycobacteria in mineral oil, Freund's incomplete adjuvant,bacterial products, such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediatedimmunity (CMI), immunogens are often emulsified in adjuvants. Manyadjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant, FCA), cytolysis (saponins andPluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

III. In Vivo DNA Vaccination.

In some preferred embodiments, nucleic acids encoding a CAIXv-GM-CSFfusion protein are incorporated into DNA vaccines. The ability ofdirectly injected DNA, that encodes an antigenic protein, to elicit aprotective immune response has been demonstrated in numerousexperimental systems (see, e.g., Conry et al. (1994) Cancer Res., 54:1164-1168; Cox et al. (1993) Virol, 67: 5664-5667; Davis et al. (1993)Hum. Mole. Genet., 2: 1847-1851; Sedegah et al. (1994) Proc. Natl. Acad.Sci., USA, 91: 9866-9870; Montgomery et al. (1993) DNA Cell Bio., 12:777-783; Ulmer et al. (1993) Science, 259: 1745-1749; Wang et al. (1993)Proc. Natl. Acad. Sci., USA, 90: 4156-4160; Xiang et al. (1994)Virology, 199: 132-140, etc.).

Vaccination through directly injecting DNA, that encodes an antigenicprotein, to elicit a protective immune response often produces bothcell-mediated and humoral responses. Moreover, reproducible immuneresponses to DNA encoding various antigens have been reported in micethat last essentially for the lifetime of the animal (see, e.g.,Yankauckas et al. (1993) DNA Cell Biol., 12: 771-776).

As indicated above, DNA vaccines are known to those of skill in the art(see, also U.S. Pat. Nos. 5,589,466 and 5,593,971, PCT/US90/01515,PCT/US93/02338, PCT/US93/04813 1, PCT/US94/00899, and the priorityapplications cited therein. In addition to the delivery protocolsdescribed in those applications, alternative methods of delivering DNAare described in U.S. Pat. Nos. 4,945,050 and 5,036,006.

Using DNA vaccine technology, plasmid (or other vector) DNA thatincludes a sequence encoding a CAIXv-GM-CSF fusion protein operablylinked to regulatory elements required for gene expression isadministered to individuals (e.g. human patients, non-human mammals,etc.). The cells of the individual take up the administered DNA and thecoding sequence is expressed. The antigen so produced becomes a targetagainst which an immune response is directed. In the present case, theimmune response directed against the antigen component of the chimericmolecule provides the prophylactic or therapeutic benefit to theindividual renal cell cancers.

The vaccines of this invention may be administered by a variety oftechniques including several different devices for administeringsubstances to tissue. The published literature includes several reviewarticles that describe aspects of DNA vaccine technology and cite someof the many reports of results obtained using the technology (see, e.g.,McDonnel and Askari (1996) New Engl. J. Med. 334(1): 42-45; Robinson(1995) Can. Med. Assoc. J. 152(10): 1629-1632; Fynan et al. (1995) Int.J Immunopharmac. 17(2): 79-83; Pardoll and Beckerleg (1995) Immunity 3:165-169; and Spooner et al. (1995) Gene Therapy 2: 173-180.

According to the present invention, the CAIXv-GM-CSF coding sequence isinserted into a plasmid (or other vector) which is then used in avaccine composition. In preferred embodiments, the CAIXv-GM-CSF codingsequence is operably linked to regulatory elements required forexpression of the construct in eukaryotic cells. Regulatory elements forDNA expression include, but are not limited to a promoter and apolyadenylation signal. In addition, other elements, such as a Kozakregion, may also be included in the genetic construct. Initiation andtermination signals are regulatory elements which are often, but notnecessarily, considered part of the coding sequence. In preferredembodiments, the coding sequences of genetic constructs of thisinvention include functional initiation and termination signals.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to, promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego, Calif.), referred toas the SV40 polyadenylation signal, may be used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to, human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

The present invention relates to methods of introducing genetic materialinto the cells of an individual in order to induce immune responsesagainst renal cell cancers. The methods comprise the steps ofadministering to the tissue of said individual, DNA that includes acoding sequence for a CAIXv-GM-CSF fusion protein operably linked toregulatory elements required for expression. The DNA can be administeredin the presence of adjuvants or other substances that have thecapability of promoting DNA uptake or recruiting immune system cells tothe site of the inoculation. It should be understood that, in preferredembodiments, the DNA transcription unit itself is expressed in the hostcell by transcription factors provided by the host cell, or provided bya DNA transcriptional unit. A DNA transcription unit can comprisenucleic acids that encode proteins that serve to stimulate the immuneresponse such as a cytokine, proteins that serve as an adjuvant andproteins that act as a receptor.

Vectors containing the nucleic acid-based vaccine of the invention canbe introduced into the desired host by methods known in the art, e.g.,transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter (see,e.g., Wu et al (1992) J. Biol. Chem. 267: 963-967; Wu and Wu (1988) J.Biol. Chem. 263: 14621-14624). The subject can be inoculatedintramuscularly, intranasally, intraperatoneally, subcutaneously,intradermally, topically, or by a gene gun.

The subject can also be inoculated by a mucosal route. The DNAtranscription unit can be administered to a mucosal surface by a varietyof methods, including lavage, DNA-containing nose-drops, inhalants,suppositories or by microsphere encapsulated DNA. For example, the DNAtranscription unit can be administered to a respiratory mucosal surface,such as the trachea or into any surface including the tongue or mucousmembrane.

The DNA transcription units are preferably administered in a medium,i.e., an adjuvant, that acts to promote DNA uptake and expression.Preferably, a pharmaceutically acceptable, inert medium is suitable asan adjuvant for introducing the DNA transcription unit into the subject.One example of a suitable adjuvant is alum (alumina gel), though even asaline solution is acceptable. Other possible adjuvants include organicmolecules such as squalines, iscoms, organic oils and fats.

An immuno-effector can be co-expressed with the CAIXv-GM-CSF nucleicacid of this present invention and thereby enhance the immune responseto the antigen. A nucleic acid encoding the immuno-effector may beadministered in a separate DNA transcription unit, operatively linked toa suitable DNA promoter, or alternatively the immuno-effector may beincluded in a DNA transcription unit comprising a nucleic acid thatencodes the CAIXv-GM-CSF construct that are operatively linked to one ormore DNA promoters. Other embodiments contain two or more suchimmuno-effectors operatively linked to one or more promoters. Thenucleic acid can consist of one contiguous polymer, encoding both thechimeric protein and the immuno-effector or it can consist ofindependent nucleic acid segments that individually encode the chimericmolecule and the immuno-effector respectively. In the latter case, thenucleic acid may be inserted into one vector or the independent nucleicacid segments can be placed into separate vectors. The nucleic acidencoding the immuno-effector and the chimeric molecule may be eitheroperatively linked to the same DNA promoter or operatively linked toseparate DNA promoters. Adding such an immuno-effector is known in theart. Alternatively, soluble immuno-effector proteins (cytokines,monokines, interferons, etc.) can be directly administered into thesubject in conjunction with the CAIXv-GM-CSF DNA.

Examples of immuno-effectors include, but are not limited to,interferon-α, interferon-γ, interferon-β, interferon-T, interferon-θ,tumor necrosis factor-a, tumor necrosis factor-β, interleukin-2,interleukin-6, interleukin-7, interleukin-12, interleukin-15, B7-1 Tcell co-stimulatory molecule, B7-2 T cell co-stimulatory molecule,immune cell adhesion molecule (ICAM)-1, T cell co-stimulatory molecule,granulocyte colony stimulatory factor, granulocyte-macrophage colonystimulatory factor, and combinations thereof.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a functioning extrachromosomal molecule and/or integrateinto the cell's chromosomal DNA. DNA may be introduced into cells whereit remains as separate genetic material, e.g., in the form of a plasmidor plasmids. Alternatively, linear DNA which can integrate into thechromosome may be introduced into the cell. When introducing DNA intothe cell, reagents which promote DNA integration into chromosomes may beadded. DNA sequences which are useful to promote integration may also beincluded in the DNA molecule. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may remain part of the genetic material inattenuated live microorganisms or recombinant microbial vectors whichlive in cells. Gene constructs may be part of genomes of recombinantviral vaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal.

Genetic constructs can be provided with a mammalian origin ofreplication in order to maintain the construct extrachromosomally andproduce multiple copies of the construct in the cell. Thus, for example,plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain theEpstein Barr virus origin of replication and nuclear antigen EBNA-1coding region which produces high copy episomal replication withoutintegration.

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the CAIXv-GM-CSFnucleic acid construct.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells intowhich the construct is administered. Moreover, codons may be selectedwhich are most efficiently transcribed in the cell. One having ordinaryskill in the art can produce DNA constructs which are functional in thecells.

The concentration of the dosage is preferably sufficient to provide aneffective immune response. The dosage of the recombinant vectorsadministered will depend upon the properties of the formulationemployed, e.g., its in vivo plasma half-life, the concentration of therecombinant vectors in the formulation, the administration route, thesite and rate of dosage, the clinical tolerance of the subject, and thelike, as is well within the skill of one skilled in the art. Differentdosages may be utilized in a series of inoculations; the practitionermay administer an initial inoculation and then boost with relativelysmaller doses of the recombinant vectors or other boosters.

The preferred dose range is between about 30 microgram to about 1 mgDNA, and more preferably between about 50 microgram to 500 microgram.Lower doses may be used as plasmid expression and inoculation areoptimized. Dosages may differ for adults in contrast to adolescents orchildren. The inoculation is preferably followed by boosters.

IV. Adoptive Immunotherapy.

Adoptive immunotherapy refers to a therapeutic approach for treatingcancer or infectious diseases in which immune cells are administered toa host with the aim that the cells mediate either directly or indirectlyspecific immunity to (i.e., mount an immune response directed against)tumor cells. In preferred embodiments, the immune response results ininhibition of tumor and/or metastatic cell growth and/or proliferationand most preferably results in neoplastic cell death and/or resorption.The immune cells can be derived from a different organism/host(exogenous immune cells) or can be cells obtained from the subjectorganism (autologous immune cells).

The immune cells are typically activated in vitro by a particularantigen (in this case CAIXv), optionally expanded, and then re-infusedback into the source organism (e.g., patient). Methods of performingadoptive immunotherapy are well known to those of skill in the art (see,e.g., U.S. Pat. Nos. 5,081,029, 5,985,270, 5,830,464, 5,776,451,5,229,115, 690,915, and the like).

In preferred embodiments, this invention contemplates numerousmodalities of adoptive immunotherapy, e.g. as described above. In oneembodiment, dendritic cells (e.g.

isolated from the patient or autologous dendritic cells) are pulsed withCAIXv or the CAIXv-GM-CSF chimeric molecule and then injected back intothe subject where they present and activate immune cells in vivo. Inaddition, or alternatively, the dentritic cells can be transfected withnucleic acids encoding the CAIXv-GM-CSF fusion protein and thenre-introduced into a patient.

In another embodiment, modified macrophage or dendritic cell (antigenpresenting cells) are pulsed with CAIXv-GM-CSF fusion proteins ortransfected with nucleic acids encoding a CAIXv-GM-CSF fusion protein,and then used to stimulate peripheral blood lymphocytes or TIL inculture and activate CAIXv-targeted CTLs that are then infused into thepatient.

Similarly, fibroblasts, and other APCs, or tumor cells (e.g. RCCs) aretransfected with a nucleic acid expressing a CAIXv-GM-CSF and used toactivate tumor cells or PBLs ex vivo to produce CAIXv directed CTLs thatcan then be infused into a patient.

Similarly various “transfection agents” including, but not limited togene therapy vectors (e.g. adenovirus, gutless-adenovirus, retrovirus,lantivirus, adeno-associated virus, vaccinia virus etc), cationiclipids, liposomes, dendrimers, and the like, containing or complexedwith a nucleic acid encoding a CAIXv-GM-CSF fusion protein areadministered to PBLs or to tumor cells (e.g. RCCs) ex vivo to produceCAIXv directed CTLs.

In one particularly preferred emobdiments, tumor cells (e.g. RCC cells)transfected to express a CAIXv-GM-CSF protein are used to provide anoff-the-shelf vaccine effective against tumors expressing a CAIXvantigen or an antigen that is cross-reactive with CAIXv.

Using the teachings provided herein, other therapeutic modalitiesutilizing CAIXv-GM-CSF polypeptides or CAIXv-GM-CSF nucleic acids can bereadily developed.

As indicated above, in one embodiment the immune cells are derived fromperipheral blood lymphocytes or TILs (e.g. derived from tumors/tumorsuspension). Lymphocytes used for in vitro activation include, but arenot limited to T lymphocytes, various antigen presenting cells (e.g.monocytes, dendritic cells, B cells, etc.) and the like. Activation caninvolve contacting an antigen presenting cell with the chimericmolecule(s) of this invention which then present the CAIXv antigen (orfragment thereof), e.g., on HLA class I molecules and/or on HLA class IImolecules, and/or can involve contacting a cell (e.g. T-lymphocyte)directly with the chimeric molecule. The antigen-presenting cells(APCs), including but not limited to macrophages, dendritic cells andB-cells, are preferably obtained by production in vitro from stem andprogenitor cells from human peripheral blood or bone marrow as describedby Inaba et al., (1992) J. Exp. Med. 176:1693-1702.

Activation of immune cells can take a number of forms. These include,but are not limited to the direct addition of the chimeric molecule toperipheral blood lymphocytes (PBLs) or tumor infiltrating lymphocytes(TILs) in culture, loading of antigen presenting cells (e.g. monocytes,dendritic cells, etc.) with the chimeric molecule in culture,transfection of antigen presenting cells, or PBLs, with a nucleic acidencoding the GM-CSF-CAIXv chimeric fusion protein, and the like.

APC can be obtained by any of various methods known in the art. In apreferred aspect human macrophages and/or dendritic cells are used,obtained from human blood donors. By way of example but not limitation,PBLs (e.g. T-cells) can be obtained as follows:

Approximately 200 ml of heparinized venous blood is drawn byvenipuncture and PBL are isolated by Ficoll-hypaque gradientcentrifugation, yielding approximately 1 to 5.times.10.sup.8 PBL,depending upon the lymphocyte count of the donor(s). The PBL are washedin phosphate-buffered saline and are suspended at approximately2.times.10.sup.5/ml in

RPMI 1640 medium containing 10% pooled heat-inactivated normal humanserum; this medium will be referred to as “complete medium.”

Similarly, other cells (e.g. mononuclear cells) are isolated fromperipheral blood of a patient (preferably the patient to be treated), byFicoll-Hypaque gradient centrifugation and are seeded on tissue culturedishes which are pre-coated with the patient's own serum or with otherAB+ human serum. The cells are incubated at 37° C. for 1 hr, thennon-adherent cells are removed by pipetting. To the adherent cells leftin the dish, is added cold (4° C.) 1 mM EDTA in phosphate-bufferedsaline and the dishes are left at room temperature for 15 minutes. Thecells are harvested, washed with RPMI buffer and suspended in RPMIbuffer. Increased numbers of macrophages may be obtained by incubatingat 37° C. with macrophage-colony stimulating factor (M-CSF); increasednumbers of dendritic cells may be obtained by incubating withgranulocyte-macrophage-colony stimulating factor (GM-CSF) as describedin detail by Inaba et al. (1992) J. Exp. Med. 176:1693-1702, and morepreferably by incubating with the CAIXv-GM-CSF chimeric molecules ofthis invention and, optionally IL-4).

The cells (e.g. APCs) are sensitized by contacting/incubating them withthe chimeric molecule. In some embodiments, sensitization may beincreased by contacting the APCs with heat shock protein(s) (hsp)noncovalently bound to the chimeric molecule. It has been demonstratedthat hsps noncovalently bound to antigenic molecules can increase APCsensitization in adoptive immunotherapeutic applications (see, e.g.,U.S. Pat. No. 5,885,270).

In one preferred embodiment, e.g. as described in the examples herein,CAIXv-GM-CSF fusion protein (with optional IL-4) is added into thepatients PBMC ex vivo and then cultured at 37.degree. C. for 7 days. Theculture is re-stimulated weekly with IL-2 and fusion protein, e.g. for 4to 5 cycles until the culture shows anti-tumor activity againstautologous kidney tumor cells displaying CAIXv. The CTLs are thenreinfused back into the patient.

For re-infusion, the cells are washed three times and resuspended in aphysiological medium preferably sterile, at a convenient concentration(e.g., 1×10⁷/ml) for injection in a patient. The cell suspension is thenfiltered, e.g., through sterile 110 mesh and put into Fenwall transferpacks. Samples of the cells are tested for the presence ofmicroorganisms including fungi, aerobic and anaerobic bacteria, andmycoplasma. A sample of the cells is optionally retained forimmunological testing in order to demonstrate induction of specificimmunity.

In a preferred embodiment, before use in immunotherapy, the stimulatedlymphocytes are tested for cell-mediated immune reactivity against tumorcells bearing the CAIXv. The PBL/TIL, following stimulation with thechimeric molecules of this invention can be examined with regard to cellsurface expression of T and B cell markers by immunofluorescent analysisusing fluorescein-conjugated monoclonal antibodies to T and B cellantigens. Expression of known T cell markers, such as the CD4 and CD8antigens, confirms the identity of the activated lymphocytes as T cells.

The activated cells (e.g. activated T cells) are then, optionally,tested for reactivity against CAIXv. This could be accomplished by anyof several techniques known in the art for assaying specificcell-mediated immunity. For example, a cytotoxicity assay, whichmeasures the ability of the stimulated T cells to kill tumor cellsbearing the CAIXv or CAIXv antigen in vitro, may be accomplished byincubating the lymphocytes with CAIXv-bearing tumor cells containing amarker (e.g. ⁵¹Cr-labelled cells) and measuring ⁵¹Cr release upon lysis.Such assays have been described (see, e.g., Zarling et al. (1986) J.Immunol. 136: 4669). The activated PBL could also be tested for T helpercell activity by measuring their ability to proliferate, as shown by³H-thymidine incorporation, following stimulation, and/or by measuringtheir ability to produce lymphokines such as IL-2 or interferon uponstimulation, in the absence of exogenous IL-2. Other assays of specificcell-mediated immunity known in the art, such as leukocyte-adherenceinhibition assays (Thomson, D. M. P. (ed.), 1982, Assessment of ImmuneStatus by the Leukocyte Adherence Inhibition Test, Academic Press, NewYork), may also be used.

Inoculation of the activated cells is preferably through systemicadministration. The cells can be administered intravenously through acentral venous catheter or into a large peripheral vein. Other methodsof administration (for example, direct infusion into an artery) arewithin the scope of the invention. Approximately 1×10⁸ cells are infusedinitially and the remainder are infused over the following severalhours. In some regimens, patients may optionally receive in addition asuitable dosage of a biological response modifier including but notlimited to the cytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF or othercytokine growth factor, antisense TGFβ, antisense IL-10, and the like.Thus, in some patients, recombinant human IL-2 may be used and will beinfused intravenously every 8 hours beginning at the time of T cellinfusion. Injections of IL-2 will preferably be at doses of 10,000 to100,000 units/kg bodyweight, as previously used in cancer patients(Rosenberg et al. (1985) N. Engl. J. Med. 313:1485). The IL-2 infusionmaybe continued for several days after infusion of the activated T cellsif tolerated by the patient.

Treatment by inoculation of, e.g., activated T cells can be used aloneor in conjunction with other therapeutic regimens including but notlimited to administration of IL-2 (as described supra), otherchemotherapeutics (e.g. doxirubicin, vinblastine, vincristine, etc.),radiotherapy, surgery, and the like.

As indicated above, the cells may, optionally, be expanded in culture.This expansion can be accomplished by repeated stimulation of the Tcells with the CAIXv-GM-CSF construct of this invention with or withoutIL-2 or by growth in medium containing IL-2 alone. Other methods of Tcell cultivation (for example with other lymphokines, growth factors, orother bioactive molecules) are also within the scope of the invention.For example, antibodies or their derivative molecules which recognizethe Tp67 or Tp44 antigens on T cells have been shown to augmentproliferation of activated T cells (Ledbetter et al. (1985) J. Immunol.135: 2331), and maybe used during in vitro activation to increaseproliferation. Interferon has been found to augment the generation ofcytotoxic T cells (Zarling et al. (1978) Immunol. 121: 2002), and may beused during in vitro activation to augment the generation of cytotoxic Tcells against CAIXv bearing cancer cells.

The description provided above details various methods for isolation,activation, and expansion of PBL. However the present invention providesfor the use CAIXv-GM-CSF constructs in various forms, and modificationsand adaptations to the method to accommodate these variations. Thusmodifications of various adoptive immunotherapeutic approaches utilizingthe CAIXv-GM-CSF constructs are within the scope of the invention.

V. Gene Transfer for Systemic Therapy or for Adoptive Immunotherapy.

In addition to use of the chimeric GM-CSF-CAIXv chimeric protein foractivation in adoptive immunotherapy, cells, (e.g., APCs, PBLs,fibroblasts, TILs, or RCC tumor cells) can be transfected with a vectorexpressing the chimeric molecule and used for adoptive immunotherapyand/or vaccine therapy.

In one preferred embodiment, the nucleic acid(s) encoding theGM-CSF-CAIXv chimeric fusion proteins are cloned into gene therapyvectors that are competent to transfect cells (such as human or othermammalian cells) in vitro and/or in vivo.

Several approaches for introducing nucleic acids into cells in vivo, exvivo and in vitro have been used. These include lipid or liposome basedgene delivery (WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite(1988) BioTechniques 6(7): 682-691; Rose U.S. Pat No. 5,279,833; WO91/06309; and Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7414) and replication-defective retroviral vectors harboring atherapeutic polynucleotide sequence as part of the retroviral genome(see, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990);Kolberg (1992) J. NIH Res. 4: 43, and Cornetta et al. (1991) Hum. GeneTher. 2: 215).

For a review of gene therapy procedures, see, e.g., Anderson, Science(1992) 256: 808-813; Nabel and Feigner (1993) TIBTECH 11: 211-217;Mitani and Caskey (1993) TIBTECH 11: 162-166; Mulligan (1993) Science,926-932; Dillon (1993) TIBTECH 11: 167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10): 1149-1154; Vigne (1995)Restorative Neurology and Neuroscience 8: 35-36; Kremer and Perricaudet(1995) British Medical Bulletin 51(1) 31-44; Haddada et al. (1995) inCurrent Topics in Microbiology and Immunology, Doerfler and Bohm (eds)Springer-Verlag, Heidelberg Germany; and Yu et al., (1994) Gene Therapy,1: 13-26.

Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiencyvirus (SIV), human immunodeficiency virus (HIV), alphavirus, andcombinations thereof (see, e.g., Buchscher et al. (1992) J. Virol.66(5)2731-2739; Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992);Sommerfelt et al., (1990) Virol. 176:58-59; Wilson et al (1989) J.Virol. 63:2374-2378; Miller et al., J. Virol. 65:2220-2224 (1991);Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) inFundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., NewYork and the references therein, and Yu et al. (1994) Gene Therapy,supra; U.S. Pat. No. 6,008,535, and the like).

The vectors are optionally pseudotyped to extend the host range of thevector to cells which are not infected by the retrovirus correspondingto the vector. For example, the vesicular stomatitis virus envelopeglycoprotein (VSV-G) has been used to construct VSV-G-pseudotyped HIVvectors which can infect hematopoietic stem cells (Naldini et al. (1996)Science 272:263, and Akkina et al. (1996) J. Virol 70:2581).

Adeno-associated virus (AAV)-based vectors are also used to transducecells with target nucleic acids, e.g., in the in vitro production ofnucleic acids and peptides, and in in vivo and ex vivo gene therapyprocedures. See, West et al. (1987) Virology 160:38-47; Carter et al.(1989) U.S. Pat. No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin(1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst.94:1351 for an overview of AAV vectors. Construction of recombinant AAVvectors are described in a number of publications, including Lebkowski,U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol.5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081; Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470; McLaughlin et al. (1988) and Samulski et al. (1989) J.Virol., 63:03822-3828. Cell lines that can be transformed by rAAVinclude those described in Lebkowski et al. (1988) Mol. Cell. Biol.,8:3988-3996. Other suitable viral vectors include herpes virus,lentivirus, and vaccinia virus.

In addition to viral vectors, a number of non-viral transfection methodsare available. Such methods include, but are not limited toelectroporation methods, calcium phosphate transfection, liposomes,cationic lipid complexes, water-oil emulsions, polethylene imines, anddendrimers.

Liposomes were first described in 1965 as a model of cellular membranesand quickly were applied to the delivery of substances to cells.Liposomes entrap DNA by one of two mechanisms which has resulted intheir classification as either cationic liposomes or pH-sensitiveliposomes. Cationic liposomes are positively charged liposomes whichinteract with the negatively charged DNA molecules to form a stablecomplex. Cationic liposomes typically consist of a positively chargedlipid and a co-lipid. Commonly used co-lipids include dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC).Co-lipids, also called helper lipids, are in most cases required forstabilization of liposome complex. A variety of positively charged lipidformulations are commercially available and many other are underdevelopment. Two of the most frequently cited cationic lipids arelipofectamine and lipofectin. Lipofectin is a commercially availablecationic lipid first reported by Phil Feigner in 1987 to deliver genesto cells in culture. Lipofectin is a mixture ofN-[1-(2,3-dioleyloyx)propyl]- —N—N—N-trimethyl ammonia chloride (DOTMA)and DOPE.

DNA and lipofectin or lipofectamine interact spontaneously to formcomplexes that have a 100% loading efficiency. In other words,essentially all of the DNA is complexed with the lipid, provided enoughlipid is available. It is assumed that the negative charge of the DNAmolecule interacts with the positively charged groups of the DOTMA. Thelipid:DNA ratio and overall lipid concentrations used in forming thesecomplexes are extremely important for efficient gene transfer and varywith application. Lipofectin has been used to deliver linear DNA,plasmid DNA, and RNA to a variety of cells in culture. Shortly after itsintroduction, it was shown that lipofectin could be used to delivergenes in vivo. Following intravenous administration of lipofectin-DNAcomplexes, both the lung and liver showed marked affinity for uptake ofthese complexes and transgene expression. Injection of these complexesinto other tissues has had varying results and, for the most part, aremuch less efficient than lipofectin-mediated gene transfer into eitherthe lung or the liver.

pH-sensitive, or negatively-charged liposomes, entrap DNA rather thancomplex with it. Since both the DNA and the lipid are similarly charged,repulsion rather than complex formation occurs. Yet, some DNA doesmanage to get entrapped within the aqueous interior of these liposomes.In some cases, these liposomes are destabilized by low pH and hence theterm pH-sensitive. To date, cationic liposomes have been much moreefficient at gene delivery both in vivo and in vitro than pH-sensitiveliposomes. pH-sensitive liposomes have the potential to be much moreefficient at in vivo DNA delivery than their cationic counterparts andshould be able to do so with reduced toxicity and interference fromserum protein.

In another approach dendrimers complexed to the DNA have been used totransfect cells. Such dendrimers include, but are not limited to,“starburst” dendrimers and various dendrimer polycations.

Dendrimer polycations are three dimensional, highly ordered oligomericand/or polymeric compounds typically formed on a core molecule ordesignated initiator by reiterative reaction sequences adding theoligomers and/or polymers and providing an outer surface that ispositively changed. These dendrimers may be prepared as disclosed inPCT/US83/02052, and U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737,4,587,329, 4,631,337, 4,694,064, 4,713,975, 4,737,550, 4,871,779,4,857,599.

Typically, the dendrimer polycations comprise a core molecule upon whichpolymers are added. The polymers may be oligomers or polymers whichcomprise terminal groups capable of acquiring a positive charge.Suitable core molecules comprise at least two reactive residues whichcan be utilized for the binding of the core molecule to the oligomersand/or polymers. Examples of the reactive residues are hydroxyl, ester,amino, imino, imido, halide, carboxyl, carboxyhalide maleimide,dithiopyridyl, and sulfhydryl, among others. Preferred core moleculesare ammonia, tris-(2-aminoethyl)amine, lysine, omithine, pentaerythritoland ethylenediamine, among others. Combinations of these residues arealso suitable as are other reactive residues.

Oligomers and polymers suitable for the preparation of the dendrimerpolycations of the invention are pharmaceutically-acceptable oligomersand/or polymers that are well accepted in the body. Examples of theseare polyamidoamines derived from the reaction of an alkyl ester of an.alpha.,.beta.-ethylenically unsaturated carboxylic acid or an.alpha.,.beta.-ethylenically unsaturated amide and an alkylene polyamineor a polyalkylene polyamine, among others. Preferred are methyl acrylateand ethylenediamine. The polymer is preferably covalently bound to thecore molecule.

The terminal groups that may be attached to the oligomers and/orpolymers should be capable of acquiring a positive charge. Examples ofthese are azoles and primary, secondary, tertiary and quaternaryaliphatic and aromatic amines and azoles, which may be substituted withS or O, guanidinium, and combinations thereof. The terminal cationicgroups are preferably attached in a covalent manner to the oligomersand/or polymers. Preferred terminal cationic groups are amines andguanidinium. However, others may also be utilized. The terminal cationicgroups may be present in a proportion of about 10 to 100% of allterminal groups of the oligomer and/or polymer, and more preferablyabout 50 to 100%.

The dendrimer polycation may also comprise 0 to about 90% terminalreactive residues other than the cationic groups. Suitable terminalreactive residues other than the terminal cationic groups are hydroxyl,cyano, carboxyl, sulfhydryl, amide and thioether, among others, andcombinations thereof. However others may also be utilized.

The dendrimer polycation is generally and preferably non-covalentlyassociated with the polynucleotide. This permits an easy disassociationor disassembling of the composition once it is delivered into the cell.Typical dendrimer polycation suitable for use herein have a molecularweight ranging from about 2,000 to 1,000,000 Da, and more preferablyabout 5,000 to 500,000 Da. However, other molecule weights are alsosuitable. Preferred dendrimer polycations have a hydrodynamic radius ofabout 11 to 60 .ANG., and more preferably about 15 to 55 .ANG. Othersizes, however, are also suitable. Methods for the preparation and useof dendrimers in gene therapy are well known to those of skill in theart and describe in detail, for example, in U.S. Pat. No. 5,661,025.

Where appropriate, two or more types of vectors can be used together.For example, a plasmid vector may be used in conjunction with liposomes.In the case of non-viral vectors, nucleic acid may be incorporated intothe non-viral vectors by any suitable means known in the art. Forplasmids, this typically involves ligating the construct into a suitablerestriction site. For vectors such as liposomes, water-oil emulsions,polyethylene amines and dendrimers, the vector and construct may beassociated by mixing under suitable conditions known in the art.

VI. Administration of GM-CSF-CAIXv with Other Agents.

In various embodiments, the GM-CSF-CAIXv fusion proteins, or nucleicacids encoding the GM-CSF-CAIXv fusion proteins can be administered inconjunction with other agents. Such agents include, but are not limitedto various chemotherapeutic agents (e.g. doxirubicin and derivatives,taxol and derivatives, vinblastine, vincristine, camptothecinderivatives, and the like, various cytokines (e.g. IL-2, IL-7, IL-12,IFN, etc.), various cytotoxins (e.g. Pseudomonas exotoxin andderivatives, diphtheria toxin and derivatives, ricin and derivatives,abrin and derivatives, thymidine kinase and derivatives), antisensemolecules (e.g. antisense IL-10, TGF-β, etc.), antibodies againstvarious growth factors/receptors (e.g. anti-VEGF, anti-EGFR, anti-IL-8,anti-FGF etc.), and the like. The methods of this invention can also beused as a adjunct to surgery, and/or radiotherapy.

VII. Kits.

Kits of the invention are provided that include materials/reagentsuseful for vaccination using a polypeptide antigen (GM-CSF-CAIXvpolypeptide) and/or DNA vaccination, and/or adoptive immunotherapy. Kitsoptimized for GM-CSF-CAIXv polypeptide vaccination preferably comprise acontainer containing a GM-CSF-CAIXv chimeric molecule. The molecule canbe provided in solution, in suspension, or as a (e.g. lyophilized)powder. The GM-CSF-CAIXv may be packaged with appropriatepharmaceutically acceptable excipient and/or adjuvant, e.g. in a unitdosage form.

Similarly, kits optimized for DNA vaccination of a construct encoding aGM-CSF-CAIXv polypeptide preferably comprise a container containing aGM-CSF-CAIXv nucleic acid (e.g. a DNA). As with the polypeptide, thenucleic acid can be provided in solution, in suspension, or as a (e.g.lyophilized) powder. The GM-CSF-CAIXv nucleic may be packaged withappropriate pharmaceutically acceptable excipient and/or facilitatingagent(s), e.g. in a unit dosage form. The kit can further includereagents and/or devices to facilitate delivery of the nucleic acid tothe subject (e.g. human or non-human mammal).

Kits optimized for adoptive immunotherapy typically include a containercontaining a chimeric GM-CSF-CAIXvpolypeptide as described above. Thekits may optionally include a nucleic acid (e.g. a vector) encoding aGM-CSF-CAIXv fusion protein for ex vivo transfection of cells. Such kitsmay also, optionally, include various cell lines (e.g. RCC) and/orreagents (e.g. IL-2) to facilitate expansion of activated cells.

The kits can, optionally, include additional reagents (e.g. buffers,drugs, cytokines, cells/cell lines, cell culture media,.etc.) and/ordevices (e.g. syringes, biolistic devices, etc.) for the practice of themethods of this invention.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. Thus typical instructional materials will teach the use ofGM-CSF-CAIXv chimeric molecules (or the nucleic acid encoding such) asvaccines, DNA vaccines, or adoptive immunotherapeutic agents in thetreatment of renal cell cancers. While the instructional materialstypically comprise written or printed materials they are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this invention. Such mediainclude, but are not limited to electronic storage media (e.g., magneticdiscs, tapes, cartridges, chips), optical media (e.g., CD ROM), and thelike. Such media may include addresses to internet sites that providesuch instructional materials.

ADDITIONAL FEATURES

The novel CAIX variant provided herein can also be used for additionalresearch, diagnostic and therapeutic purposes. Specific blockers ofCAIXv and/or high-affinity agonists for CAIXv can be exploited as noveltherapeutic agents. In addition, even more potent and specificGMCSF-CAIXv based therapies (for example, therapies targeting RCC) canbe achieved using a nanotechnology platform.

The novel CAIXv cDNA/amino acidic sequences unreported previouslyprovide a novel mechanism and signaling pathway for RCC with significantprognostic and therapeutic capabilities, as well as the promisingpotential with regard to develop new pharmacological agents andstrategies to target RCC and other CAIXv-expressing cancers.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety, to the extent notinconsistent with the present disclosure, for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

INFORMAL SEQUENCE LISTING human CAIXv full sequence SEQ ID NO: 1 MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLMPVHPQRLPRMQEDSPLGGGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVEAPGGPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQLAAFCPALRPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGRPGSEHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLSRLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAKQLHTLSDTLWGPGDSRLQLSFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAGDILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYR PAEVAETGASEQ ID NO: 2: human CAIXv nucleic acid sequenceATGGCTCCCCTGTGCCCCAGCCCCTGGCTCCCTCTGTTGATCCCGGCCCCTGCTCCAGGCCTCACTGTGCAACTGCTGCTGTCACTGCTGCTTCTGATGCCTGTCCATCCCCAGAGGTTGCCCCGGATGCAGGAGGATTCCCCCTTGGGAGGAGGCTCTTCTGGGGAAGATGACCCACTGGGCGAGGAGGATCTGCCCAGTGAAGAGGATTCGCCCAGAGAGGAGGATCCACCCGGAGAGGAGGATCTACCTGGAGAGGAGGATCTACCTGGAGAGGAGGATCTACCTGAAGTTAAGCCTAAATCAGAAGAAGAGGGCTCCCTGAAGTTAGAGGATCTACCTACCGTTGAGGCTCCTGGAGGTCCTCAAGAACCCCAGAATAATGCCCACAGGGACAAAGAAGGGGATGACCAGAGTCATTGGCGCTATGGAGGCGACCCGCCCTGGCCCCGGGTGTCCCCAGCCTGCGCGGGCCGCTTCCAGTCCCCGGTGGATATCCGCCCCCAGCTCGCCGCCTTCTGCCCGGCCCTGCGCCCCCTGGAACTCCTGGGCTTCCAGCTCCCGCCGCTCCCAGAACTGCGCCTGCGCAACAATGGCCACAGTGTGCAACTGACCCTGCCTCCTGGGCTAGAGATGGCTCTGGGTCCCGGGCGGGAGTACCGGGCTCTGCAGCTGCATCTGCACTGGGGGGCTGCAGGTCGTCCGGGCTCGGAGCACACTGTGGAAGGCCACCGTTTCCCTGCCGAGATCCACGTGGTTCACCTCAGCACCGCCTTTGCCAGAGTTGACGAGGCCTTGGGGCGCCCGGGAGGCCTGGCCGTGTTGGCCGCCTTTCTGGAGGAGGGCCCGGAAGAAAACAGTGCCTATGAGCAGTTGCTGTCTCGCTTGGAAGAAATCGCTGAGGAAGGCTCAGAGACTCAGGTCCCAGGACTGGACATATCTGCACTCCTGCCCTCTGACTTCAGCCGCTACTTCCAATATGAGGGGTCTCTGACTACACCGCCCTGTGCCCAGGGTGTCATCTGGACTGTGTTTAACCAGACAGTAATGCTGAGTGCTAAGCAGCTCCACACCCTCTCTGACACCCTGTGGGGACCCGGTGACTCTCGGCTACAACTGAGCTTCCGAGCGACGCAGCCTTTGAATGGGCGAGTGATTGAGGCCTCCTTCCCTGCTGGAGTGGACAGCAGTCCTCGGGCTGCTGAGCCAGTCCAGCTGAATTCCTGCCTGGCTGCTGGTGACATTCTAGCCCTGGTTTTTGGCCTCCTTTTTGCTGTCACCAGCGTCGCGTTCCTTGTGCAGATGAGAAGGCAGCACAGAAGGGGAACCAAAGGGGGTGTGAGCTACCGCCCAGCAGAGGTAGCCGAGACTGGAGCCTAG SEQ ID NO: 3 NCBILOCUS BC014950 1639 bp mRNA linear PRI 15-JUL-2006 DEFINITIONHomo sapiens carbonic anhydrase IX, mRNA (cDNA clone MGC:22967 IMAGE:4865275), complete cds. ACCESSION BC014950VERSION BC014950.1 GI:15928967 Human CAIX amino acid sequence   1maplcpspwl pllipapapg ltvqlllsll llmpvhpqrl prmqedsplg ggssgeddpl  61geedlpseed spreedppge edlpgeedlp geedlpevkp kseeegslkl edlptveapg 121dpqepqnnah rdkegddqsh wryggdppwp rvspacagrf qspvdirpql aafcpalrpl 181ellgfqlppl pelrlrnngh svqltlppgl emalgpgrey ralqlhlhwg aagrpgseht 241veghrfpaei hvvhlstafa rvdealgrpg glavlaafle egpeensaye qllsrleeia 301eegsetqvpg ldisallpsd fsryfqyegs lttppcaqgv iwtvfnqtvm lsakqlhtls 361dtlwgpgdsr lqlnfratqp lngrvieasf pagvdsspra aepvqlnscl aagdilalvf 421gllfavtsva flvqmrrqhr rgtkggvsyr paevaetga SEQ ID NO: 4LOCUS AAI13925 144 aa linear PRI 29-JUN-2006 DEFINITIONColony stimulating factor 2 (granulocyte-macrophage) [Homo sapiens].ACCESSION AAI13925 VERSION AAI13925.1 GI:109731225 DBSOURCEaccession BC113924.1 human GMCSF amino acid sequence   1mwlqsllllg tvacsisapa rspspstqpw ehvnaiqear rllnlsrdta aemnetvevi  61semfdlqept clqtrlelyk qglrgsltkl kgpltmmash ykqhcpptpe tscatqtitf 121esfkenlkdf llvipfdcwe pvqeStrausberg,R.L. et al., Proc. Natl. Acad. Sci. U.S.A. 99 (26), 16899-16903(2002)--

1-36. (canceled)
 37. A method of treating an individual having a cancercharacterized by an altered expression of a CAIX variant protein(CAIXv), said method comprising: (a) sensitizing antigen presentingcells in vitro with a sensitizing-effective amount of a chimeric fusionprotein comprising the CAIX variant protein attached to a granulocytemacrophage colony stimulating factor (GM-CSF), wherein the CAIX variantprotein comprises SEQ ID NO: 1 or a fragment of at least 20 contiguousamino acids of SEQ ID NO:1 that comprises G121 and S374; and (b)administering to an individual having said cancer or a metastasisthereof a therapeutically effective amount of the sensitized antigenpresenting cells.
 38. The method of claim 37, wherein the antigenpresenting cells are autologous to the individual.
 39. The method ofclaim 37, wherein said sensitizing comprises contacting cells selectedfrom the group consisting of peripheral blood lymphocytes, monocytes,fibroblasts, tumor infiltrating lymphocytes (TILs), and dendritic cellswith said chimeric fusion protein.
 40. The method of claim 37, whereinsaid sensitizing comprises transfecting dendritic cells or renal cellcarcinoma cells (RCCs) with a nucleic acid encoding said chimeric fusionprotein. 41-46. (canceled)
 47. The method of claim 37, wherein theGM-CSF is a human GM-CSF.
 48. The method of claim 37, wherein the CAIXvariant protein comprises SEQ ID NO:1.
 49. The method of claim 37,wherein the CAIX variant protein and the GM-CSF are joined by a peptidelinker ranging in length from 2 to about 20 amino acids.
 50. The methodof claim 49, wherein the peptide linker is -Arg-Arg- or -Lys-Leu-. 51.The method of claim 37, wherein the cancer is renal cell carcinoma,cervical cancer, or bladder cancer.
 52. The method of claim 51, whereinthe cancer is renal cell carcinoma.
 53. The method of claim 37, whereinthe antigen presenting cells are major histocompatibility complex(MHC)-matched allogenic dendritic cells.
 54. The method of claim 37,wherein subsequent to the sensitizing step, the method further comprisesexpanding the sensitized antigen presenting cells in culture.
 55. Themethod of claim 54, wherein the expanding step comprises contacting thesensitized antigen presenting cells with a cytokine.
 56. The method ofclaim 55, wherein the cytokine is IL-2, interferon α, or interferon γ.57. The method of claim 37, wherein the sensitized antigen presentingcells are administered systemically.
 58. The method of claim 37, whereinthe sensitized antigen presenting cells are administered intravenously.59. The method of claim 37, wherein the sensitized antigen presentingcells are administered in conjunction with a chemotherapeutic drug.